Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer&#39;s disease

ABSTRACT

The invention provides unique therapeutic and diagnostic antibodies, as well as their fragments, portions, derivatives, and variants thereof, that bind regions of the tau protein that contribute to the initiation and propagation of pathological tau-tau interactions, as well as methods of making them. The invention also relates to methods of using those antibodies for diagnostics, prevention, and treatment of Alzheimer&#39;s disease and related tauopathies. The present invention also provides a method for a prophylactic and therapeutic treatment of Alzheimer&#39;s disease and other neurodegenerative tauopathies. This method entails the injection of antibodies and/or peptide vaccines that elicits an immune response directed to pathological tau proteins and tau deposits in the brains of patients. Suitable vaccines represent a tau peptide carrying one or more of the tau therapeutic epitopes provided herein.

This claims the benefit of priority of U.S. Provisional Applications61/536,339, filed on Sep. 19, 2011, and 61/653,115, filed on May 30,2012, the content of both of which is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 13, 2012, isnamed SEQUENCE_LISTING.txt and is 155,400 bytes in size.

FIELD

The present invention features protein-based (e.g., antibodies,peptides) methods and means for interfering with the production andclearance of certain forms of tau that are involved in the promotionand/or development of pathological tau-tau aggregates in Alzheimer'sdisease, as well as methods for producing anti-tau antibodies that areuseful for diagnosis and treatment of Alzheimer's disease. The inventionfurther concerns methods and means for diagnosing Alzheimer's disease,including methods for staging and evaluating treatment progression.

BACKGROUND

Alzheimer's disease (AD) is a progressive neurodegenerative disorderthat destroys higher brain structures, such as those involved in memoryand cognition. The disease leads to deficits in cognitive function anddeclines in memory, learning, language, and in the ability to performintentional and purposeful movements. AD is also accompanied byconcomitant behavioral, emotional, interpersonal, and socialdeterioration. These cognitive and behavioral deficits render livingdifficult (Burns et al., 2002). Late-stage AD patients are often unableto speak, comprehend language, and handle their own basic personal care,eventually requiring full-time care and supervision, and are oftendependent on family members and nursing homes. AD is the leading causeof senile dementia, and is predicted to increase in prevalence as theproportion of elderly persons in the population grows. The total numberof persons with AD is predicted to increase at least threefold justbetween 2000 and 2050, rendering AD a world-wide public health problem(Sloane et al., 2002). Clinical management of AD remains largelysupportive. That is, patients are given treatments aimed at prevention,control, or relief of complications and side effects from AD, and toimprove their comfort and quality of life. There is still an unmet needfor treatments that directly target the disease process and havedisease-modifying effects.

AD is histologically characterized by the presence of extraneuronalplaques and intracellular and extracellular neurofibrillary tangles inthe brain. Plaques are composed mainly of β amyloid (Aβ), whereastangles comprise pathological forms of tau, such as pathological tauconformers and their aggregates. The relationship between plaques andtangles and the disease process remains unclear, although studiessuggest a link between amyloid and tau pathogenesis (Hardy et al., 1998;Oddo et al., 2004; Rapoport et al., 2002; Roberson, et al., 2007;Shipton et al., 2011). A central role for Aβ in AD pathology wasinitially proposed in a hypothesis called the “Aβ cascade,” wherein Aβdeposition is followed by tau phosphorylation and tangle formation, andthen neuronal death (Hardy and Allsop, 1991; Hardy and Selkoe, 2002; fora review see, Walsh and Selkoe, 2004; also see Seabrook et al. 2007).Accordingly, initial therapeutic approaches for AD focused primarily ontargeting Aβ. However, there is a documented lack of correlation betweenthe extent of brain Aβ pathology in AD patients and clinical progressionof the disease (Braak and Braak, 1991). In addition, asymptomaticindividuals have shown extensive, often diffuse, amyloid deposition atautopsy (Braak and Braak, 1991), and at least in early-stage AD,neuronal loss and amyloid deposition occur in different regions of thebrain (Carter and Lippa, 2001). Therefore targeting AR alone cannotsuffice to alter the disease process in any or all patients.Nevertheless, the most advanced disease-targeting therapies undergoingclinical trials in AD patients remain those aimed at the production andclearance of Aβ. These therapies include passive immunotherapies, e.g.,BAPINEUZUMAB, SOLANEUZUMAB, and PONEZUMAB, as well as the small moleculegamma-secretase inhibitor SEMAGACESTAT (for review see Citron et al.,2010).

A recognized role for tau in AD pathology has been demonstrated innumerous studies. For example, Braak showed that the closest correlatefor AD neurodegeneration was the presence of tau tangles, and not ofamyloid plaques (Braak and Braak, 1991). In another study, Aβneurotoxicity in cultured neurons appeared to depend on tau (Rapoport etal., 2002). Recently, reducing endogenous tau prevented behavioraldeficits in transgenic mice that expressed the human amyloid precursorprotein, without altering their high Aβ levels (Roberson et al., 2007).Tau reduction also protected both transgenic and nontransgenic miceagainst excitotoxicity. Id. Santacruz et al. demonstrated that areduction in the amount of tau restored memory function in a model oftauopathy (Santacruz et al., 2005). Thus, therapies aimed at reducingtau can represent an effective strategy for treating AD and othertau-related disease conditions.

Tau belongs to a family of intrinsically disordered proteins,characterized by the absence of a rigid three-dimensional structure intheir physiological environment (Zilka et al., 2008) However, tautruncation and hyperphosphorylation can cause pathologicaltransformations from an intrinsically disordered state to multiplesoluble and insoluble misdisordered structures, including paired helicalfilaments (PHFs) and other aggregates (Wischik et al., 1988a; Wischik etal., 1988b; Novak et al., 1993; Skrabana et al., 2006; Zilka et al.,2008; Kovacech et al., 2010). These structural changes lead to a toxicgain of function, to a loss of physiological function of the nativeprotein, or both (Zilka et al., 2008; Kovacech et al., 2010).

Tau's physiological function is in mediating the assembly of tubulinmonomers into microtubules that constitute the neuronal microtubulesnetwork (Buee et al., 2000). Tau binds to microtubules throughrepetitive regions located in the C-terminal portion of the protein. Id.These repeat domains (R1-R4), are not identical to each other, butcomprise highly conserved 31-32 amino acids (Taniguchi et al., 2005b).In the human brain, there are six unique isoforms of tau, which differfrom each other in the presence or absence of certain amino acids in theN-terminal portion of tau, in combination with either three (R1, R3, andR4) or four (R1-R4) repeat domains, at the C-terminal end of theprotein. See also FIG. 1, which shows the six human isoforms (2N4R, 1N4R, 2N3R, 0N4R, 1 N3R, and 0N3R). It has been proposed that the mostpotent part of tau to induce microtubule polymerization is the274-KVQIINKK-281 region (SEQ ID NO: 113), overlapping R1-R2. Id. Inaddition, tau's pathological and physiological functions appear to beinfluenced by the specific structural conformation, and theintrinsically disordered structure, adopted by the full length proteinisoforms and their fragments. For example, Kontsekova et al. described aconformational region (encompassing residues297-IKHVPGGGSVQIVYKPVDLSKVTSKCGSL-325 (SEQ ID NO: 114)) within certaintruncated tau molecules which had a significant relationship to thefunction of those truncated tau molecules on microtubule assembly (WO2004/007547).

In addition to their physiological role, tau repeats are believed toparticipate in the formation of pathological tau aggregates and otherstructures. Thus, there is a need for tau-targeted therapeutic anddiagnostic approaches that are capable of discriminating betweenphysiological and pathological repeat-mediated activities. For example,the pronase resistant core of pathological paired helical filaments(PHFs) consists of the microtubule binding regions of 3- and 4-repeattau isoforms (Jakes et al., 1991; Wischik, et al. 1988a; Wischik, et al.1988b). Further, Novak et al. showed that the protease resistant core ofthe PHFs, which is 93-95 amino acids long, was restricted to threetandem repeats (Novak et al., 1993). Von Bergen et al. determined aminimal-tau peptide/interaction motif (306-VQIVYK-311; SEQ ID NO: 115),as well as a second site on tau (275-VQIINK-280) (SEQ ID NO: 116), whichform beta-sheets and are described as potentially responsible forinitiating the formation of PHFs, a pathological tau aggregate (VonBergen et al., 2000; EP 1214598; WO 2001/18546). See FIG. 2 for afunctional map of tau. Consequently, current strategies aim atgenerating anti-aggregating drugs that do not disrupt tau'sintracellular role in microtubule stabilization.

Moreover, while under physiological circumstances tau is considered anintracellular cytoplasmic protein, intracellular tau can be releasedinto the extracellular space and contribute to neurodegeneration(Gómez-Ramos et al., 2006). Indeed, neuronal loss has been linked to thetopographic distribution of neurofibrillary tangles (made up of tauprotein) in AD brains (West et al., 1994; Gomez-Isla et al., 1996,1997). Further, the levels of total tau and phosphorylated tau areincreased in the cerebrospinal fluid (CSF) of patients with AD (Hampelet al., 2010), and extracellular tau has been described as “ghosttangles” in the brain (Frost and Diamond, 2009), indicating thatintracellular tau is released into extracellular space. In addition,extracellular tau aggregates can enter cells and stimulatefibrillization of intracellular tau, further seeding tau monomer forproduction of pathological tau aggregates (Frost et al., 2009). Suchstudies have highlighted that extracellular, insoluble tau could act asa transmissible agent to spread tau pathology throughout the brain in aprion-like fashion (Frost et al., 2009; Frost and Diamond, 2009).Clearance of extracellular tau tangles can reduce tau-associatedextracellular and intracellular pathology. See, e.g., Asuni et al.,2007. Therefore, there is a need for treatments capable of decreasingextracellular tau, either by impeding its formation, promoting itsclearance, or both, as well as for treatments that decreaseintracellular disease tau.

All in all, although tau appears to play a pathological role in theclinical manifestation of AD, the development of drugs that work againsttau has been slow, in part due to tau's importance in physiologicmicrotubule dynamics and to its complex biology (Dickey and Petrucelli,2006). However, an increased understanding of the molecular mechanismsunderlying the pathological transformations of tau has opened up thepossibility of specifically targeting pathological modifications of taufor therapeutic purposes. As a result, a number of therapeuticapproaches that directly or indirectly target the tau cascade haveemerged (for review articles, see, e.g. Dickey and Petrucelli, 2006;Schneider and Mandelkow, 2008; Zilka et al., 2008), including compoundsthat prevent or reverse tau aggregation (Wischik et al., 1996; Necula etal. 2005; Pickhardt et al., 2005; Taniguchi et al., 2005a; Larbig etal., 2007) small-molecule type drugs that inhibit tau kinases oractivate tau phosphatases (Iqbal and Grundke-Iqbal, 2004; Noble et al.,2005; Iqbal and Grundke-Iqbal, 2007), microtubule stabilizing drugs(Zhang et al., 2005), drugs that facilitate the proteolytic degradationof misfolded tau proteins (Dickey et al., 2005, Dickey et al. 2006;Dickey and Petrucelli, 2006), and immunosuppresive drugs (Zilka et al.,2008), as well as immunotherapeutic strategies including active andpassive immunization (Schneider and Mandelkow et al., 2008; Zilka etal., 2008: Tabira, T. Immunization Therapy for Alzheimer disease: AComprehensive Review of Active Immunization Strategies. Tohoku J. Exp.Med., 220: 95-106 (2010)).

More generally, novel monoclonal antibodies (mAbs) have been enteringclinical studies at a rate of over 40 per year since 2007. At the end of2010, at least 25 mAbs and five Fc fusion proteins were in Phase 2/3 orPhase 3 clinical studies in the US (Reichert, 2011). This trenddemonstrates that passive immunotherapy is a growing approach in thetreatment of human disorders, including AD. See, e.g., Citron et al.,2010. In fact, although AD treatments face the hurdle of overcoming theblood-brain-barrier (BBB), a growing number of pre-clinical and clinicalstudies report that antibody-mediated therapies can clear AD aggregatesfrom the brain, and propose multiple mechanisms of action, such as (i)antibody uptake into the brain via an altered BBB permeability in AD, orBBB leakage; (ii) antibodies working as “peripheral sinks” for solubleplaque-forming amyloid species; (iii) entrance of antibody-secretingcells from the periphery into the brain, delivering antibodies locally;and (iv) transport of IgG within and across cells. See, e.g., Citron etal., 2010, and Asuni et al., 2007, for review. Accordingly, therapeuticantibodies targeting disease forms of tau represent a prospectiveapproach for treatment and/or diagnosis of AD and other tauopathies (WO2004/007547, US2008/0050383).

One of the immunotherapy approaches to target tau pathology is based onthe notion that anti-tau antibodies could prevent tau aggregation, cleartau aggregates, or both. Although studies have described antibodies thatbind to tau sequences, and some of those antibodies reportedly interferewith tau aggregation and clearance (Asuni et al., 2007), no monoclonalanti-tau antibody is yet reportedly undergoing in vivo pre-clinical orclinical trials in AD. Indeed, one mAb was predicted to have threebinding sites within murine tau's microtubule-binding domain (namely, atR3, R4, and possibly R1), but it did not block microtubule binding.(Dingus et al., 1991). Dingus did not describe a role for this antibodyon tau aggregation and thus, there is no reason to believe that theDingus will block tau aggregation. In other reports, mAbs were generatedthat distinguish tau isoforms, but again there is no suggestion thatthese will have any effect on tau aggregation (DeSilva et al., 2003;Ueno et al., 2007). Taniguchi et al. demonstrated that certain anti-taumAbs against R1 or R2, inhibited tau aggregation into PHFs in vitro,while promoting tau-induced tubulin assembly (Taniguchi et al., 2005b).Taniguchi's RTA-1 and RTA-2 antibodies bound specifically to R1 and R2,respectively. Neither antibody bound more than one tau repeat and nonewas reportedly tested for in vivo effects on either tau aggregation orclearance. Despite the existence of at least three anti-amyloidantibodies in clinical trials for passive immunization-based therapy ofAD (i.e., one in which antibodies are administered to the patient), noclinical testing reports of passive, tau-based immunotherapies for ADare yet available.

An active immunization approach (i.e., one in which the patient's bodyitself generates immunity against the target) was found to be effectivein clearing Aβ deposits and reversing neuropathological lesions inseveral APP-transgenic mouse studies of AD (see, e.g. Schenk et al.,1999; Janus et al., 2000; Morgan et al., 2000; Sigurdsson et al., 2001).Recently, active immunotherapy with a phosphorylated tau epitope (Tau379-408 [P-Ser 396, 404]) reduced the extent of aggregated tau in thebrain and slowed the progression of the behavioral phenotype in mousemodels of tau tangle pathology (Asuni et al., 2007; Boutajangout et al.2010; US2008/0050383; US/2010/00316564). Treated animals producedanti-tau antibodies, which were detected in the brain and colocalizedwith antibodies that recognized pathological tau (Asuni et al., 2007).This immunotherapeutic approach was substantially more effective in theearly stages of functional impairments in the animals (5 months) than atlater stages (8 months), suggesting that clearance of early-stagepathological tau can be of therapeutic benefit (Asuni et al., 2007;Zilka et al., 2008). Indeed, there is awareness that not all tau issusceptible or perhaps even suitable for disruption and clearance. Somehave suggested that disrupting tau aggregates could increase theabundance of toxic intermediate species, while others have suggestedthat detectable tau aggregates are not necessarily toxic and can evenplay a protective role (Lee et al., 2005). Thus, althoughimmunotherapeutic approaches to target tau have shown pre-clinicalpromise, there is still a need for therapeutics that specifically targetearly, aberrant forms of tau whose elimination produces improved,lasting benefits. Nevertheless, there is still also a need to identifythose tau species that are suitable targets for immunotherapy.

To this end, another consideration for developing mAbs against tau isthe identification and characterization of the various structural formsof tau (physiological, early disease, late disease) and the stages oftau pathology that are targeted. Oddo et al. observed that while Aβimmunotherapy cleared Aβ plaques and early tau pathology in a transgenicmouse model of AD, mature tau aggregates remained intact (Oddo et al.,2004). Similarly, a genetic (not immunotherapeutic) reduction of tauexpression in a P301L tau model of tauopathy improved memory, eventhough neurofibrillary tangles continued to accumulate (Santacruz etal., 2005).

Notwithstanding its prevalence, AD remains the largest unmet medicalneed in neurology (Citron, 2010). The most prevalent medical approach isto provide symptomatic therapy, which is not efficacious even afterseveral years of treatment. New therapeutic approaches and strategiesfor AD need to go beyond the treatment of symptoms to prevent cognitivedecline and counteract the fundamental pathological processes of thedisease. In particular, there is a need for the development of moleculesthat either alone or in combination with other AD-targeted drugsinterfere with at least some of the earliest stages of the disease. Suchmolecules would provide new, advantageous options in the early diagnosis(which could itself improve treatment outcomes), prevention, andtreatment of AD.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides an isolated antibody, whereinthe antibody binds to one or more tau epitopes and is capable of two ormore of the following:

-   -   a) displaying a higher affinity for pathological tau than for        physiological tau;    -   b) inhibiting tau-tau aggregation; and    -   c) mediating uptake and degradation of pathological tau protein        by microglia;    -   and wherein each tau epitope comprises an aggregation-promoting        region of tau.

In an embodiment, this isolated antibody is such that each of the one ormore epitopes is independently selected from epitopes within:

-   -   i. position 267-273 or residues KHQPGGG (SEQ ID NO: 98),        relative to tau₄₄₁;    -   ii. position 298-304 or residues KHVPGGG (SEQ ID NO: 99),        relative to tau₄₄₁    -   iii. position 329-335 or residues HHKPGGG (SEQ ID NO: 100),        relative to tau₄₄₁; and    -   iv. position 361-367 or residues THVPGGG (SEQ ID NO: 101),        relative to tau₄₄₁.

In certain embodiments, the isolated antibody having the propertiesdescribed in the embodiments of the previous paragraphs is capable ofbinding to one or more forms of pathological tau chosen from misorderedtau, misdisordered tau, sarkosyl-insoluble tau, neurofibrillary tangles,neuropil threads, and neuritic plaques in a brain biopsy of a humanAlzheimer's disease patient, in a brain sample from an animal model ofAlzheimer's disease, or in both. In certain embodiments, the isolatedantibody is such that at least one of the epitopes that it recognizes isa conformational epitope.

In one embodiment, the invention provides an isolated antibody, whereinthe antibody binds to one or more tau epitopes and is capable of two ormore of the following:

-   -   a) displaying a higher affinity for pathological tau than for        physiological tau;    -   b) inhibiting tau-tau aggregation; and    -   c) mediating uptake and degradation of pathological tau protein        by microglia;    -   and wherein each tau epitope comprises an aggregation-promoting        region of tau.

In an embodiment, this isolated antibody is such that each of the one ormore epitopes is independently selected from epitopes within:

-   -   i. position 268-273 or residues HQPGGG (SEQ ID NO: 223),        relative to tau₄₄₁;    -   ii. position 299-304 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁    -   iii. position 330-335 or residues HKPGGG (SEQ ID NO: 224),        relative to tau₄₄₁; and    -   iv. position 362-367 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁.

In an embodiment, this isolated antibody is such that each of the one ormore epitopes it binds to is independently selected from epitopeswithin:

-   -   i. position 268-273 or residues HQPGGG (SEQ ID NO: 223),        relative to tau₄₄₁;    -   ii. position 299-304 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁    -   iii. position 330-335 or residues HKPGGG (SEQ ID NO: 224),        relative to tau₄₄₁; and    -   iv. position 362-367 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁;    -   and the antibody comprises:    -   a) an antibody light chain variable region comprising:        -   i. QSLLNSRTRKNY (SEQ ID NO: 117) or SEQ ID NO: 247 for CDR1;        -   ii. WAS (SEQ ID NO: 118) or SEQ ID NO: 253 for CDR2; and        -   iii. KQSFYLRT (SEQ ID NO: 119) or any one of SEQ ID NOs:            255, 257, 258, 259, and 260 for CDR3; and    -   b) an antibody heavy chain variable region comprising:        -   iv. GYIFTDYVIS (SEQ ID NO: 120), SEQ ID NO: 261, or SEQ ID            NO: 262 for CDR1;        -   v. IFPRSGST (SEQ ID NO: 121), SEQ ID NO: 264, or SEQ ID NO:            265 for CDR2; and        -   vi. ARDYYGTSFAMDY (SEQ ID NO: 122), SEQ ID NO: 266, SEQ ID            NO: 267, or SEQ ID NO: 269 for CDR3.

The invention also provides an isolated antibody that binds one or moreepitopes on tau in a conformationally-specific manner wherein:

-   -   a) each of the one or more epitopes is independently selected        from epitopes within:        -   i. position 267-273 or residues KHQPGGG (SEQ ID NO: 98),            relative to tau₄₄₁;        -   ii. position 298-304 or residues KHVPGGG (SEQ ID NO: 99),            relative to tau₄₄₁        -   iii. position 329-335 or residues HHKPGGG (SEQ ID NO: 100),            relative to tau₄₄₁; and        -   iv. position 361-367 or residues THVPGGG (SEQ ID NO: 101),            relative to tau₄₄₁,    -   b) zero, one, two, or three of the epitopes is/are linear        epitope(s); and    -   c) one, two, three, or four of the epitopes is/are        conformational epitope(s).

The invention also provides an isolated antibody that binds one or moreepitopes on tau in a conformationally-specific manner wherein:

-   -   a) each of the one or more epitopes is independently selected        from epitopes within:        -   i. position 268-273 or residues HQPGGG (SEQ ID NO: 223),            relative to tau₄₄₁;        -   ii. position 299-304 or residues HVPGGG (SEQ ID NO: 154),            relative to tau₄₄₁        -   iii. position 330-335 or residues HKPGGG (SEQ ID NO: 224),            relative to tau₄₄₁; and        -   iv. position 362-367 or residues HVPGGG (SEQ ID NO: 154),            relative to tau₄₄₁.    -   b) zero, one, two, or three of the epitopes is/are linear        epitope(s); and    -   c) one, two, three, or four of the epitopes is/are        conformational epitope(s).

In one embodiment, this antibody is DC8E8, wherein DC8E8 is an antibodyproduced by the hybridoma deposited under American Type CultureCollection Patent Deposit no. PTA-11994.

In certain embodiments, the isolated antibody binds to one or more ofthe same epitopes on tau as those bound by DC8E8. In an embodiment, theisolated antibody competes with monoclonal antibody DC8E8 for binding totau.

The invention also provides an isolated antibody comprising in itsepitope binding domain one or more complementarity determining region(CDR) sequences chosen from:

(SEQ ID NO: 117) i. QSLLNSRTRKNY (SEQ ID NO: 118) ii. WAS(SEQ ID NO: 119) iii. KQSFYLRT (SEQ ID NO: 120) iv. GYIFTDYVIS(SEQ ID NO: 121) v. IFPRSGST; and (SEQ ID NO: 122) vi. ARDYYGTSFAMDY.

The invention also provides that any of the antibodies described in anyembodiments described in the preceding paragraphs can be such that theisolated antibody comprises:

-   -   a) an antibody light chain variable region comprising:        -   i. QSLLNSRTRKNY (SEQ ID NO: 117) for CDR1;        -   ii. WAS (SEQ ID NO: 118) for CDR2; and        -   iii. KQSFYLRT (SEQ ID NO: 119) for CDR3; and    -   b) an antibody heavy chain variable region comprising:        -   iv. GYIFTDYVIS (SEQ ID NO: 120) for CDR1        -   v. IFPRSGST (SEQ ID NO: 121) for CDR2; and        -   vi. ARDYYGTSFAMDY (SEQ ID NO: 122) for CDR3.

The invention also provides that any of the antibodies described in theprevious embodiments can be such that the isolated antibody comprises:

-   -   a) one or more sequences of the light chain CDRs from the        monoclonal antibody DC8E8, or one or more sequences having at        least 80%, 90%, or 95% identity after optimum alignment with one        of these light chain CDRs; and    -   b) one or more sequences of the heavy chain CDRs from the        monoclonal antibody DC8E8, or one or more sequences having at        least 80%, 90%, or 95% identity after optimum alignment with one        of these heavy chain CDRs;        and wherein:    -   i. the light chain CDRs comprise a sequence chosen from        QSLLNSRTRKNY (SEQ ID NO: 117), WAS (SEQ ID NO: 118), and        KQSFYLRT (SEQ ID NO: 119); and    -   ii. the heavy chain CDRs comprise a sequence chosen from        GYIFTDYVIS (SEQ ID NO: 120), IFPRSGST (SEQ ID NO: 121), and        ARDYYGTSFAMDY (SEQ ID NO: 122).

The invention also provides that any of the antibodies described in theprevious embodiments can consist of or comprise a Fab, Fab′, F(ab′)₂,Fabc, Fv fragment, any other antigen-binding fragment; or anantigen-binding antibody portion thereof; having one or more of thefollowing immunological binding characteristics:

-   -   1. the antibody binds one or more tau epitopes in a        conformationally-specific manner, wherein:        -   a) each of the one or more tau epitopes is independently            selected from epitopes within:            -   i. position 267-273 or residues KHQPGGG (SEQ ID NO: 98),                relative to tau₄₄₁;            -   ii. position 298-304 or residues KHVPGGG (SEQ ID NO:                99), relative to tau₄₄₁            -   iii. position 329-335 or residues HHKPGGG (SEQ ID NO:                100), relative to tau₄₄₁; and            -   iv. position 361-367 or residues THVPGGG (SEQ ID NO:                101), relative to tau₄₄₁;        -   b) zero, one, two, or three of the epitopes is a linear            epitope;        -   c) one, two, three, or four of the epitopes is a            conformational epitope;    -   2. the antibody binds two or more tau epitopes and is capable of        displaying a higher affinity for pathological tau than for        physiological tau, wherein the two tau epitopes are selected        from epitopes within:        -   v. position 267-273 or residues KHQPGGG (SEQ ID NO: 98),            relative to tau441;        -   vi. position 298-304 or residues KHVPGGG (SEQ ID NO: 99),            relative to tau441        -   vii. position 329-335 or residues HHKPGGG (SEQ ID NO: 100),            relative to tau441; and        -   viii. position 361-367 or residues THVPGGG (SEQ ID NO: 101),            relative to tau441.

The invention also provides that any of the antibodies described in theprevious embodiments can consist of or comprise a Fab, Fab′, F(ab′)₂,Fabc, Fv fragment, any other antigen-binding fragment; or anantigen-binding antibody portion thereof; having one or more of thefollowing immunological binding characteristics:

-   -   1. the antibody binds one or more tau epitopes in a        conformationally-specific manner, wherein:        -   a) each of the one or more tau epitopes is independently            selected from epitopes within:            -   i. position 268-273 or residues HQPGGG (SEQ ID NO: 223),                relative to tau₄₄₁;            -   ii. position 299-304 or residues HVPGGG (SEQ ID NO:                154), relative to tau₄₄₁            -   iii. position 330-335 or residues HKPGGG (SEQ ID NO:                224), relative to tau₄₄₁; and            -   iv. position 362-367 or residues HVPGGG (SEQ ID NO:                154), relative to tau₄₄₁.        -   b) zero, one, two, or three of the epitopes is a linear            epitope;        -   c) one, two, three, or four of the epitopes is a            conformational epitope;    -   2. the antibody binds two or more tau epitopes and is capable of        displaying a higher affinity for pathological tau than for        physiological tau, wherein the two tau epitopes are selected        from epitopes within:        -   i. position 268-273 or residues HQPGGG (SEQ ID NO: 223),            relative to tau₄₄₁;        -   ii. position 299-304 or residues HVPGGG (SEQ ID NO: 154),            relative to tau₄₄₁        -   iii. position 330-335 or residues HKPGGG (SEQ ID NO: 224),            relative to tau₄₄₁; and        -   iv. position 362-367 or residues HVPGGG (SEQ ID NO: 154),            relative to tau₄₄₁.

The invention also relates to any isolated antibody that competitivelybinds to tau against any of the isolated antibodies described in theprevious embodiments. In one embodiment, the isolated antibodycompetitively binds to tau when tested against isolated DC8E8 forbinding to tau.

In some embodiments, the antibody comprises a light chain comprising SEQID NO.: 141. In some embodiments, the antibody comprises a light chaincomprising SEQ ID NO.:138. In some embodiments, the antibody comprises alight chain comprising SEQ ID NO.: 141 and a light chain comprising SEQID NO.:138.

The invention provides that the antibodies provided by the invention canbe chosen from:

-   -   a) a monoclonal antibody;    -   b) a polyclonal antibody;    -   c) a recombinant antibody;    -   d) a chimeric antibody;    -   e) a humanized antibody;    -   f) a human antibody; and    -   g) an antigen-binding fragment or antigen-binding portion of        anyone of (a) through (f).

Any of the isolated antibodies provided by the invention can be raisedin a mammal. In certain embodiments, the isolated antibody is producedby a recombinant animal or by a recombinant host cell.

The invention provides that any of the isolated anti-tau antibodiesprovided herein can be such that they are detectably labeled with one ormore labeling agents. In certain embodiments, at least one labelingagent is chosen from an enzyme, a radioisotope, a fluorophore, a nuclearmagnetic resonance marker, and a heavy metal.

In some embodiments, the antibody comprises at least one drug(combination agent) attached to the antibody molecule.

The invention also provides isolated nucleic acids encoding at least oneCDR, or at least the binding domain or variable region of animmunoglobulin chain of any of the anti-tau antibodies described in theprevious embodiments. Also provided are isolated vectors comprising anyof those nucleic acids. In some embodiments, the invention provides anisolated host cell comprising one or more of these isolated nucleicacids and vectors.

In certain embodiments, the invention provides an isolated cell lineexpressing any of the anti-tau antibodies described in the previousembodiments. In one embodiment, the isolated cell line is a hybridoma.In one embodiment, the isolated cell line is the hybridoma from whichmonoclonal antibody DC8E8 is produced, and which cell line has beendeposited with the American Type Culture Collection, Manassas, Va., USA,on Jul. 13, 2011, with the ATCC Patent Deposit Designation PTA-11994.

The invention provides for the use of any of the anti-tau antibodies,nucleic acids, and cells provided herein, as a drug or in themanufacture of a medicament for the diagnosis, prevention, or treatmentof Alzheimer's disease or a related tauopathy.

In some embodiments, the antibodies are comprised in a pharmaceuticalcomposition, further comprising pharmaceutically acceptable carrierand/or diluent. In one embodiment, the pharmaceutical compositioncomprises a combination of antibodies and a pharmaceutically acceptablecarrier and/or diluent, wherein the combination comprises at least twodifferent antibodies, and wherein each of the antibodies isindependently selected from the antibodies described in the previousembodiments. In one embodiment, at least one of the antibodies is DC8E8,or a human version of DC8E8, or a humanized version of DC8E8.

In some embodiments, the antibodies are comprised in a composition,further comprising a diluent and/or a carrier. The composition can be apharmaceutical composition, a diagnostic composition, or any othercomposition. In some embodiments, the composition can further compriseat least one compound or agent selected from a detectable label, keyholelimpet hemocyanin, tetanus toxoid or a toxoid derived from otherpathogenic bacteria, serum albumins, bovine serum albumin, animmunoglobulin molecule or fragment thereof, thyroglobulin, ovoglobulin,a universal T-cell epitope, a cytokine, a chemokine, interleukin 1-alpha(IL-1α), IL-1β, IL-2, IL-10, interferon-gamma (IFN-γ), granulocytemacrophage colony-stimulating factor (GM-CSF), macrophage inflammatoryprotein 1 alpha (MIP1α), MIP1β, and RANTES (regulated upon activation,normal T-cell expressed and secreted).

The invention also provides an article of manufacture (e.g., a kit) forpharmaceutical or diagnostic use, comprising packaging material and acontainer comprising a solution of a lyophilized form any one or more ofthe anti-tau antibodies provided herein. In certain embodiments, thecontainer is a component of a device or system for delivery of theantibody to a subject.

In some embodiments, the invention provides a medical device comprisingan anti-tau antibody as provided herein (see above), wherein the deviceis suitable for contacting or administering the antibody by at least onemode selected from parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracerebellar,intracerebroventricular, intrathecal, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, intralesional,bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

In one embodiment, the invention relates to a method of treating orpreventing the progression of Alzheimer's disease or a related tauopathyin a subject, the method comprising administering to said subject aneffective amount of at least one of the anti-tau antibodies providedherein. In some embodiments, the method is capable of reducing motorimpairment, improving motor function, reducing cognitive impairment,improving cognitive function, or of a combination thereof.

In certain embodiments, the invention relates to a method ofameliorating at least one of the symptoms associated with Alzheimer'sdisease or a related tauopathy in a subject, the method comprisingadministering to said subject an effective amount of at least one of theanti-tau antibodies provided herein.

In still another embodiment, the invention provides a method ofdiagnosing or screening a subject for the presence of Alzheimer'sdisease or a related tauopathy in a subject, or for determining asubject's risk for developing Alzheimer's disease or a relatedtauopathy, the method comprising:

-   -   a) contacting the subject, or a cell, tissue, organ, fluid, or        any other sample of the subject, with an effective amount of at        least one anti-tau antibody as provided herein; and    -   b) determining the presence of a complex comprising pathological        tau and the antibody, wherein the presence of the complex is        diagnostic of Alzheimer's disease or a related tauopathy        associated with the presence of pathological tau.

In a related embodiment, the invention provides a method of monitoring asubject for the presence, progression, regression, or stabilization ofAlzheimer's disease or a related tauopathy in a subject, or fordetermining the stage of Alzheimer's disease or a related tauopathy in asubject, for the method comprising:

-   -   a) contacting the subject, or a cell, tissue, organ, fluid, or        any other sample of the subject, with an effective amount of at        least one of the anti-tau antibodies provided herein; and    -   b) determining the presence and/or characteristics of a complex        comprising pathological tau and the antibody, wherein the        presence of the complex is diagnostic of Alzheimer's disease or        a related tauopathy associated with the presence of pathological        tau.

In some embodiments, the antibody is administered intravenously,intramuscularly, subcutaneously, intraperitoneally, intranasally,intracerebroventricularly, intrathecally, or as an aerosol.

In some embodiments of the methods of treating or preventing theprogression of Alzheimer's disease or a related tauopathy in a subject,and of the methods of ameliorating at least one of the symptomsassociated with Alzheimer's disease or a related tauopathy in a subject,the effective amount of each antibody is at least 1 mg/kg body weight ofthe subject, per dose. In some embodiments, the effective amount of eachantibody is at least 10 mg/kg body weight of the subject, per dose. Insome embodiments, at least one of the antibodies is administered inmultiple dosages over a period of at least six months. In someembodiments, the antibody is administered peripherally to a humansubject to exert its beneficial effects. In some embodiments, theantibody, when administered peripherally to a human subject, binds tosoluble tau, sarkosyl-insoluble tau, or to both. In some embodiments,the antibody, when administered peripherally to a human subject, bindsto tau, wherein tau is in one or more pathological forms chosen frommisordered tau, misdisordered tau, sarkosyl-insoluble tau,neurofibrillary tangles, neuropil threads, and neuritic plaques in abrain biopsy of a human Alzheimer's disease patient, in a brain samplefrom an animal model of Alzheimer's disease. In some embodiments, theantibody, when administered peripherally to a human subject, exerts oneor more effector-function mediated beneficial effects on the subject. Insome embodiments, the antibody is delivered to the periphery byinjection/implantation of an antibody-expressing cell into the subject'sbrain. In some embodiments, the antibody-expressing cell is an hybridomacell. In some embodiments, the hybridoma cell is a hybridoma expressingDC8E8.

In certain related embodiments, the invention provides an isolatedpeptide, wherein:

-   -   a) the isolated peptide is a fragment of tau that is at least 6        amino-acid-residues-long, at least 7 amino-acid-residues-long,        at least 9 amino-acid-residues-long, at least 10        amino-acid-residues-long, at least 12 amino-acid-residues-long,        or 30 amino-acid-residues-long; and    -   b) the isolated peptide comprises a tau therapeutic epitope.

In some related embodiments, the therapeutic epitope comprises atherapeutic epitope selected from those within:

-   -   i. position 267-273 or residues KHQPGGG (SEQ ID NO: 98),        relative to tau₄₄₁;    -   ii. position 298-304 or residues KHVPGGG (SEQ ID NO: 99),        relative to tau₄₄₁    -   iii. position 329-335 or residues HHKPGGG (SEQ ID NO: 100),        relative to tau₄₄₁; and    -   iv. position 361-367 or residues THVPGGG (SEQ ID NO: 101),        relative to tau₄₄₁.

In certain related embodiments, the invention provides an isolatedpeptide, wherein:

-   -   a) the isolated peptide is a fragment of tau that is at least 6        amino-acid-residues-long, at least 7 amino-acid-residues-long,        at least 9 amino-acid-residues-long, at least 10        amino-acid-residues-long, at least 12 amino-acid-residues-long,        or 30 amino-acid-residues-long; and    -   b) the isolated peptide comprises a tau therapeutic epitope.

In some related embodiments, the therapeutic epitope comprises atherapeutic epitope selected from those within:

-   -   i. position 268-273 or residues HQPGGG (SEQ ID NO: 223),        relative to tau₄₄₁;    -   ii. position 299-304 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁    -   iii. position 330-335 or residues HKPGGG (SEQ ID NO: 224),        relative to tau₄₄₁; and    -   iv. position 362-367 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁.

In some related embodiments, the therapeutic epitope is selected from:

-   -   i. position 268-273 or residues HQPGGG (SEQ ID NO: 223),        relative to tau₄₄₁;    -   ii. position 299-304 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁    -   iii. position 330-335 or residues HKPGGG (SEQ ID NO: 224),        relative to tau₄₄₁; and    -   iv. position 362-367 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁.

In other embodiments, the isolated peptide is a sequence selected fromSEQ ID NOs: 1-4, SEQ ID NOs: 9-101, and SEQ ID NOs: 108-112, NIKAVPGGGS(SEQ ID NO: 200), NIKHVPGGGS (SEQ ID NO: 201), IKHVPGGGS (SEQ ID NO:202), KHVPGGGSV (SEQ ID NO: 203), HVPGGGSVQ (SEQ ID NO: 204), VPGGGSVQ(SEQ ID NO: 205), GWSIHSPGGGSC (SEQ ID NO: 250), SVFQHLPGGGSC (SEQ IDNO: 251), ANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS (SEQ ID NO: 146),DNAKHVPGGGS (SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO: 151), DNIKAVPGGGS(SEQ ID NO: 159), DNIKHAPGGGS (SEQ ID NO: 161), and DNIKHVPGGGS (SEQ IDNO: 171).

In other embodiments, the isolated peptide is a sequence selected fromSEQ ID NO: 270 (TENLKHQPGGGK); SEQ ID NO: 271 (KHQPGGG), SEQ ID NO: 272(HQPGGG); SEQ ID NO: 275 (ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS),SEQ ID NO: 276 (KHVPGGG), SEQ ID NO: 277 (HVPGGG), SEQ ID NO: 280(DNIKHVPGGGSVQIVYKPV), SEQ ID NO: 281 (HHKPGGG), SEQ ID NO: 282(HKPGGG), and SEQ ID NO: 283 (THVPGGG).

In other embodiments, the isolated peptide is a sequence selected fromSEQ ID NO: 270 (TENLKHQPGGGK); SEQ ID NO: 271 (KHQPGGG), SEQ ID NO: 272(HQPGGG); SEQ ID NO: 275 (ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS),SEQ ID NO: 276 (KHVPGGG), SEQ ID NO: 277 (HVPGGG), SEQ ID NO: 280(DNIKHVPGGGSVQIVYKPV), SEQ ID NO: 281 (HHKPGGG), SEQ ID NO: 282(HKPGGG), and SEQ ID NO: 283 (THVPGGG); and the therapeutic epitope isselected from:

-   -   i. position 268-273 or residues HQPGGG (SEQ ID NO: 223),        relative to tau₄₄₁;    -   ii. position 299-304 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁    -   iii. position 330-335 or residues HKPGGG (SEQ ID NO: 224),        relative to tau₄₄₁; and    -   iv. position 362-367 or residues HVPGGG (SEQ ID NO: 154),        relative to tau₄₄₁.

In other embodiments, the isolated peptide is a sequence selected fromSEQ ID NO: 272 (HQPGGG) and SEQ ID NO: 277 (HVPGGG).

In certain embodiments, the isolated peptide is active in at least oneassay, selected from assays that measure the peptide's:

-   -   a) ability to compete with tau for binding to the monoclonal        antibody DC8E8;    -   b) ability to reduce the level of sarkosyl-insoluble tau, in        vivo;    -   c) ability to promote tau clearance from the brain, in vivo;    -   d) ability to reduce the level of at least one biochemical        marker of AD, in vivo;    -   e) ability to reduce neurofibrillary tangle (NFT) load, in vivo;    -   f) ability to improve at least one neurobehavioral parameter, in        vivo;    -   g) ability to beneficially modify the course of AD in a subject;    -   h) ability to reduce the level of tau in the brain, in the        cerebrospinal fluid, or in both; and/or    -   i) ability to serve as an immunogen in the making of an antibody        capable of competing with monoclonal DC8E8 for binding to tau.

The invention also relates to compounds comprising any of the isolatedpeptides provided herein and a moiety. In certain embodiments, themoiety is N-terminal, C-terminal, or linked to an internal amino acid ofthe peptide, and wherein the moiety is selected from one or more of acysteine residue, phospho group, keyhole limpet hemocyanin, tetanustoxoid or a toxoid derived from other pathogenic bacteria, serumalbumins, bovine serum albumin, an immunoglobulin molecule or fragmentthereof, thyroglobulin, ovoglobulin, a universal T-cell epitope, acytokine, a chemokine, interleukin 1-alpha (IL-1α), IL-1β, IL-2, IL-10,interferon-gamma (IFN-γ), granulocyte macrophage colony-stimulatingfactor (GM-CSF), macrophage inflammatory protein 1 alpha (MIP1α), MIP1β,and RANTES (regulated upon activation, normal T-cell expressed andsecreted).

Also provided are pharmaceutical compositions comprising one or more ofthe isolated peptides and/or compounds provided by the invention and apharmaceutically acceptable carrier, and/or a diluent, and/or anadjuvant. In some embodiments, the pharmaceutical composition is adaptedto provide a dosage of the peptide or of the compound between 1 ng and10 mg. In certain embodiments, the pharmaceutical composition is adaptedto provide a dosage of the peptide or of the compound greater than 10micrograms.

The invention also relates to an article of manufacture (e.g., a kit)for pharmaceutical or diagnostic use, comprising packaging material anda container comprising a solution of a lyophilized form of a peptideand/or compound provided by the invention. In some embodiments, thecontainer is a component of a device or system for delivery of thepeptide or the compound to a subject.

Also provided are medical devices comprising a peptide, a compound,and/or a peptide/compound composition as provided by the invention,wherein the device is suitable for contacting or administering theantibody by at least one mode selected from parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intrathecal,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual,intranasal, and transdermal.

In related embodiments, the invention provides a method of treating orpreventing the progression of Alzheimer's disease or related tauopathiesin a subject, the method comprising administering to said subject aneffective amount of at least one peptide and/or at least one compound asprovided by the invention. In some embodiments, the method is capable ofreducing motor impairment, improving motor function, reducing cognitiveimpairment, improving cognitive function, or a combination thereof.

In related embodiments, the invention provides a method of amelioratingat least one of the symptoms associated with Alzheimer's disease orrelated tauopathies in a subject, the method comprising administering tosaid subject an effective amount of at least one peptide and/or at leastone compound as provided by the invention.

In some of these methods of treatment, prevention, or amelioration of atleast one of the symptoms associated with a method of ameliorating atleast one of the symptoms associated with Alzheimer's disease or arelated tauopathy in a subject, the method comprises administering to ahuman patient a peptide and/or a compound as provided by the invention,and/or an adjuvant that augments the immune response, which methodeffects an immune response comprising antibodies against pathologicaltau, thereby treating, preventing the progression, or ameliorating atleast one of the symptoms associated with AD in the human patient.

The invention also provides a method of producing an antibody that isable to compete with DC8E8 for binding to tau, the method comprisingimmunizing a subject with at least one peptide and/or with at least onecompound as provided by the invention. In some embodiments, at least onepeptide is a peptide is chosen from any one of SEQ ID NOs: 1-4, SEQ IDNOs: 9-101, and SEQ ID NOs: 108-112, NIKHVPGGGS (SEQ ID NO: 201),IKHVPGGGS (SEQ ID NO: 202), KHVPGGGSV (SEQ ID NO: 203), HVPGGGSVQ (SEQID NO: 204), VPGGGSVQ (SEQ ID NO: 205), GWSIHSPGGGSC (SEQ ID NO: 250),SVFQHLPGGGSC (SEQ ID NO: 251), ANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS(SEQ ID NO: 146), DNAKHVPGGGS (SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO:151), DNIKAVPGGGS (SEQ ID NO: 159), DNIKHAPGGGS (SEQ ID NO: 161), andDNIKHVPGGGS (SEQ ID NO: 171). In one embodiment, the peptide is chosenfrom SEQ ID NOs: 1-4. In another embodiment, the peptide is SEQ ID NO.108. In one embodiment, the peptide is GWSIHSPGGGSC (SEQ ID NO: 250). Incertain embodiments, the peptide is SVFQHLPGGGSC (SEQ ID NO: 251). Incertain embodiments the peptide is selected from SEQ ID NO: 270(TENLKHQPGGGK); SEQ ID NO: 271 (KHQPGGG), SEQ ID NO: 272 (HQPGGG); SEQID NO: 275 (ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS), SEQ ID NO: 276(KHVPGGG), SEQ ID NO: 277 (HVPGGG), SEQ ID NO: 280(DNIKHVPGGGSVQIVYKPV), SEQ ID NO: 281 (HHKPGGG), SEQ ID NO: 282(HKPGGG), and SEQ ID NO: 283 (THVPGGG). In other embodiments, thepeptide is selected from SEQ ID NO: 272 (HQPGGG) and SEQ ID NO: 277(HVPGGG).

Also provided is a method of isolating DC8E8, or isolating an antibodythat is able to compete with DC8E8 for binding to tau, the methodcomprising contacting DC8E8 or the antibody with a peptide and/or with acompound as provided by the invention.

In related embodiments, the invention provides a method of diagnosing orscreening a subject for the presence of Alzheimer's disease or relatedtauopathies in a subject, or for determining a subject's risk fordeveloping Alzheimer's disease or related tauopathies, the methodcomprising:

-   -   a) contacting the subject, or a cell, tissue, organ, fluid, or        any other sample of the subject, with an effective amount of at        least one antibody as provided by the invention; and    -   b) determining the presence of a complex comprising pathological        tau and the antibody, wherein the presence of the complex is        diagnostic of Alzheimer's disease or related tauopathies        associated with the presence of pathological tau.

In certain embodiments, the invention provides a method of monitoring asubject for the presence, progression, regression, or stabilization ofAlzheimer's disease or related tauopathies, or for determining the stageof Alzheimer's disease or related tauopathies in a subject, the methodcomprising:

-   -   a) contacting (e.g., administering) the subject, or a cell,        tissue, organ, fluid, or any other sample of the subject, with        an effective amount of at least one antibody as provided by at        least one embodiment of the invention; and    -   b) determining the presence and/or characteristics of a complex        comprising pathological tau and the antibody, wherein the        presence of the complex is diagnostic of Alzheimer's disease or        related tauopathies associated with the presence of pathological        tau.

In some embodiments of the method of monitoring a subject for thepresence, progression, regression, or stabilization of Alzheimer'sdisease or related tauopathies, or for determining the stage ofAlzheimer's disease or related tauopathies in a subject, the antibody,peptide, and/or compound is administered intravenously, intramuscularly,subcutaneously, intraperitoneally, intranasally,intracerebroventricularly, intrathecally, or as an aerosol. In someembodiments, the effective amount of each peptide and/or compound is atleast 1 μg per dose, at least 10 μg per dose, at least 100 μg per dose.In some embodiments, the effective amount of each peptide and/orcompound is at least 10 μg per dose in the presence of an adjuvant, andat least 100 μg per dose in the absence of an adjuvant. In someembodiments, at least one peptide or compound is administered inmultiple dosages over a period of at least six months.

According to a related embodiment, the invention provides a method oftreating or preventing the progression of Alzheimer's disease or relatedtauopathies in a subject, the method comprising administering to saidsubject an effective amount of at least one antibody, and/or at leastone peptide, and/or at least one compound as provided by the invention,in combination with at least one combination agent chosen fromacetylcholinesterase inhibitors, N-Methyl-D-aspartate (NMDA) receptorantagonists, transition metal chelators, growth factors, hormones,non-steroidal anti-inflammatory drugs (NSAID), antioxidants, lipidlowering agents, selective phosphodiesterase inhibitors, inhibitors oftau aggregation, inhibitors of protein kinases, inhibitors of heat shockproteins, anti-amyloid passive and active immunization, anti-amyloidaggregation inhibitors, and secretase inhibitors. In some embodiments,the method is capable of reducing motor impairment, improving motorfunction, reducing cognitive impairment, improving cognitive function,or a combination thereof.

In a related embodiment, the invention provides a method of amelioratingat least one of the symptoms associated with Alzheimer's disease orrelated tauopathies in a subject, the method comprising administering tosaid subject an effective amount of at least one antibody, at least onepeptide, and/or at least one compound as provided by the invention, incombination with at least one combination agent chosen fromacetylcholinesterase inhibitors, NMDA receptor antagonists, transitionmetal chelators, growth factors, hormones, non-steroidalanti-inflammatory drugs (NSAID), antioxidants, lipid lowering agents,selective phosphodiesterase inhibitors, inhibitors of tau aggregation,inhibitors of protein kinases, inhibitors of heat shock proteins,anti-amyloid-passive and -active immunization reagents, anti-amyloidaggregation inhibitors, and secretase inhibitors.

In some embodiments of the methods of treatment, prevention, oramelioration of at least one of the symptoms associated with Alzheimer'sdisease or related tauopathies in a subject, the method comprisesadministering to a human patient an effective amount of at least oneantibody, at least one peptide, and/or at least one compound as providedby the invention, and/or an adjuvant that augments the immune response;in combination with at least one combination agent chosen fromacetylcholinesterase inhibitors, NMDA receptor antagonists, transitionmetal chelators, growth factors, hormones, non-steroidalanti-inflammatory drugs (NSAID), antioxidants, lipid lowering agents,selective phosphodiesterase inhibitors, inhibitors of tau aggregation,inhibitors of protein kinases, inhibitors of heat shock proteins,anti-amyloid passive and -active immunization, anti-amyloid aggregationinhibitors, and secretase inhibitors; wherein the method effects animmune response comprising antibodies against pathological tau, therebytreating, preventing the progression, or ameliorating at least one ofthe symptoms associated with AD in the human patient.

In some embodiments of the methods of treatment, prevention, oramelioration of at least one of the symptoms associated with Alzheimer'sdisease or related tauopathies in a subject, the combination agent isadministered prior to, simultaneously with, or after the administrationof an antibody, a peptide, and/or a compound as provided by theinvention.

In a related embodiment, the invention also provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and/ordiluent; and

-   -   a) an antibody as provided by the invention; and/or    -   b) a peptide as provided by the invention; and/or    -   c) a compound as provided by the invention;    -   in combination with at least one combinantion agent chosen from        acetylcholinesterase inhibitors, NMDA receptor antagonists,        transition metal chelators, growth factors, hormones,        non-steroidal anti-inflammatory drugs (NSAID), antioxidants,        lipid lowering agents, selective phosphodiesterase inhibitors,        inhibitors of tau aggregation, inhibitors of protein kinases,        inhibitors of heat shock proteins, anti-amyloid-passive and        -active immunization reagents, anti-amyloid aggregation        inhibitors, and secretase inhibitors. In some embodiments, the        antibody is DC8E8. In certain embodiments, the antibody        comprises at least one CDR from DC8E8. In some embodiments, the        antibody comprise at least one variable chain (light or heavy)        from DC8E8. In certain embodiments, a humanized or human version        of DC8E8 can be used. In some embodiments, at least one peptide        is chosen from any one of SEQ ID NOs: 1-4, SEQ ID NOs: 9-101,        and SEQ ID NOs: 108-112, NIKHVPGGGS (SEQ ID NO: 201), IKHVPGGGS        (SEQ ID NO: 202), KHVPGGGSV (SEQ ID NO: 203), HVPGGGSVQ (SEQ ID        NO: 204), VPGGGSVQ (SEQ ID NO: 205), GWSIHSPGGGSC (SEQ ID NO:        250), SVFQHLPGGGSC (SEQ ID NO: 251), ANIKHVPGGGS (SEQ ID NO:        144), DAIKHVPGGGS (SEQ ID NO: 146), DNAKHVPGGGS (SEQ ID NO:        149), DNIAHVPGGGS (SEQ ID NO: 151), DNIKAVPGGGS (SEQ ID NO:        159), DNIKHAPGGGS (SEQ ID NO: 161), and DNIKHVPGGGS (SEQ ID NO:        171). In one embodiment, the peptide is chosen from SEQ ID NOs:        1-4. In another embodiment, the peptide is SEQ ID NO. 108. In        one embodiment, the peptide is GWSIHSPGGGSC (SEQ ID NO: 250). In        certain embodiments, the peptide is SVFQHLPGGGSC (SEQ ID NO:        251). In certain embodiments the peptide is selected from SEQ ID        NO: 270 (TENLKHQPGGGK); SEQ ID NO: 271 (KHQPGGG), SEQ ID NO: 272        (HQPGGG); SEQ ID NO: 275        (ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS), SEQ ID NO: 276        (KHVPGGG), SEQ ID NO: 277 (HVPGGG), SEQ ID NO: 280        (DNIKHVPGGGSVQIVYKPV), SEQ ID NO: 281 (HHKPGGG), SEQ ID NO: 282        (HKPGGG), and SEQ ID NO: 283 (THVPGGG). In other embodiments,        the peptide is selected from SEQ ID NO: 272 (HQPGGG) and SEQ ID        NO: 277 (HVPGGG).

Additional objects and advantages of the embodiments will be set forthin part in the description which follows, and in part will be obviousfrom the description, or can be learned by practice of the embodiments.The objects and advantages of the embodiments will be realized andattained by means of the elements and combinations particularly pointedout in the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the embodiments, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description, serve to explain the principles of theembodiments. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.They are all incorporated by reference in their entirety for allpurposes. Nothing herein is to be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

Further, the dates of publication provided can be different from theactual publication dates which can need to be independently confirmed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic of six isoforms of human tau.

FIG. 2: Schematic functional map of human tau (2N4R). FIG. 2 discloses“VQIINK” and “VQIVYK” as SEQ ID NOS 116 and 115, respectively.

FIG. 3: The nucleotide and amino-acid sequences of DC8E8 variableregions and their alignment to the closest mouse germ line sequences.The figure shows nucleotide (SEQ ID NO: 165) (A) and amino acid (SEQ IDNOS 141 (for the variable light chain) and 117-119 (for each of itsCDRs, according to IMGT), respectively, in order of appearance); (B)sequences of the variable light (VL) chain region of DC8E8 (alignmentdiscloses SEQ ID NOS 166 and 168, respectively, in order of appearance);and (C) alignment of DC8E8's variable light chain V-gene to the closestmouse germline sequence IGKV8-21*01 (alignment discloses SEQ ID NOS 166and 167, respectively, in order of appearance; followed by the alignmentof DC8E8's VL J-gene (SEQ ID NO:168) to closest mouse J gene, IGKJ1*01(SEQ ID NO: 169). The figure shows the nucleotide (SEQ ID NO: 170) (inD) and amino acid sequence of DC8E8's variable heavy chain and its threeCRDs (SEQ ID NOS 171 and 120-122, respectively, in order of appearance)sequences. In (F) are shown the following alignments for DC8E8: first,the variable heavy (VH) chain V-gene of DC8E8 (SEQ ID NO 172) with theclosest mouse germline sequence IGHV1-8101 (SEQ ID NO 172); second, thevariable heavy (VH) chain D-gene of DC8E8 (SEQ ID NO 174) with theclosest mouse germline sequence IGHD2-14*01 (SEQ ID NO 175); and last,the variable heavy (VH) chain J-gene of DC8E8 (SEQ ID NO 176) with theclosest mouse germline sequence IGHJ4*01 (SEQ ID NO 177). The sequenceof DC8E8 kappa light chain constant region (SEQ ID NO: 178) (G) andsequence of heavy chain constant region (SEQ ID NO: 179) (H) are alsoshown. Complementarity determining regions (CDRs) are underlined in theprotein sequences (B) and (E) and were identified according to IMGTnumbering system.

FIG. 4: Alignment of DC8E8 Variable Light (VL) chain sequence (SEQ IDNOS 166 (V-gene) and 168 (J-gene), respectively, to the closest humangermline VL gene (SEQ ID NOS 180-181, respectively, in order ofappearance).

FIG. 5: Alignment of DC8E8 Variable Heavy (VH) chain sequence (SEQ IDNOS 172,174, and 176, for V, D, and J genes, respectively) to theclosest human germline VH gene (SEQ ID NOS 182-183 and 185,respectively, in order of appearance).

FIG. 6: Epitope mapping of DC8E8 by tau deletion mutants using ELISA.(A) Schematic of tau proteins used for DC8E8 epitope mapping, and (B)their amino acid sequence (SEQ ID NOS 186-197, 102, 104, and 198-199,respectively, in order of appearance). (C) ELISA readouts. DC8E8recognizes the following tau proteins: Δ358-441, Δ421-441, Δ134-168,Δ1-220, Δ1-126, 2N4R, 2N3R, Δ(1-296;392-441) and Δ(1-150;392-441)/4R.DC8E8 does not recognize the following tau proteins: Δ222-427, Δ306-400,Δ228-441, Δ300-312, Δ257-400, Δ137-441, Δ283-441.

FIG. 7: (A) and (B) Schematic of synthetic peptides (SEQ ID NOS 206,207, 208, 2, 210, 211, 212, 3, 214, 215, 4, 217, 26, 219, 36, 221, 222,109, and 88, respectively, in order of appearance) for epitope mappingand their sequence, respectively. (C) Epitope mapping of DC8E8 withsynthetic peptide by ELISA. (D) Schematic of epitopes that DC8E8 iscapable of binding to within tau. DC8E8 is capable of binding to any oneof four separate binding regions, each of which is a separate epitope,named epitope 1 through 4. The four epitopes each are separately locatedwithin the 1^(st) (epitope #1), 2^(nd) (epitope #2), 3rd (epitope #3),and 4^(th) (epitope #4) repeat domains of protein tau. As shown, thefour DC8E8 epitopes are each respectively encompassed within one of eachof the following amino acid sequences: 267-KHQPGGG-273 (SEQ ID NO: 98)(within 1^(st) repeat domain of tau protein), 298-KHVPGGG-304 (SEQ IDNO: 99) (within 2^(nd) repeat domain of tau protein), 329-HHKPGGG-335(SEQ ID NO: 100) (within 3^(rd) repeat domain of tau protein) and361-THVPGGG-367 (SEQ ID NO: 101) (within 4^(th) repeat domain of tauprotein), respectively.

FIG. 8: (A) Alignment of human tau amino acid sequence (SEQ ID NO: 225)to tau protein sequence from other species (SEQ ID NOS 226-245,respectively, in order of appearance). The full length of human tauprotein was used for the alignment; only amino acids 265-368 of humantau from the alignment are shown. The regions comprising the fourseparate DC8E8 epitopes on human tau and the aligned sequences are boxedand shown in bold. (B) Competition ELISA showing the ability of six taupeptides (SEQ ID NOS 201-205 and 200, respectively, in order ofappearance) to compete with tauΔ(1-150;392-441)/4R (SEQ ID NO: 199) forbinding to antibody DC8E8, capable (C) Competition ELISA showing theability of seven tau peptides (SEQ ID NOS 144, 146, 149, 151, 159, 161,and 171) to compete with tauΔ(1-150;392-441)/4R for binding to antibodyDC8E8, capable of recognizing at least one of the tau epitopes involvedin tau-tau aggregation of recognizing at least one of the tau epitopesinvolved in tau-tau aggregation.

FIG. 9: (A). Surface plasmon resonance (SPR) to characterize DC8E8'sbinding to tauΔ(1-150;392-441)/4R and 2N4R. (B). Surface plasmonresonance (SPR) to characterize DC8E8's binding totauΔ(1-150;392-441)/3R and 2N3R.

FIG. 10: (A). Association and dissociation rates of DC8E8 binding totauΔ(1-150;392-441)/4R and to tau 2N4R, as determined by SPR. (B).Association and dissociation rates of DC8E8 binding totauΔ(1-150;392-441)/3R and to tau 2N3R, as determined by SPR. Theconcentrations used in the measurements are indicated in the plots,dashed lines were interpolated by computer program BIA evaluationsoftware 4.1 (Biacore AB) from measured data for kinetic parametercalculations.

FIG. 11: Monoclonal antibody DC8E8 is able to discriminate betweenpreclinical AD, clinically incipient AD and fully developed final stageAD. DC8E8 displays staining of early stages (tau monomers, dimers) ofpathological tau in human preclinical AD—Braak's Stage I. (A). Theantibody recognizes the stage of pathological tau oligomers (arrows) andthe stage of pathological tau polymers (tangles) (arrowhead) (B). Infully developed Alzheimer's disease (final stage—Braak's Stage VI),DC8E8 recognizes mainly pathological tau polymers in forms of theneurofibrillary tangles (arrowhead), neuritic plaques (inside thecircle) and neuritic threads (inside the pentagon) (C). Scale bar: 100μm. Monoclonal antibody DC8E8 recognizes all developmental stages oftangle formation in Alzheimer's disease (D). DC8E8 recognizes earlydevelopmental stages of tangle formation—monomeric, dimeric and earlyoligomeric stage (D1), and late oligomeric, pre-tangle stage (D2), aswell as late developmental stages of pathological taupolymers—intracellular (D3) and extracellular neurofibrillary tangles(D4). Arrowhead indicates small oligomeric tau aggregates insidepyramidal hippocampal neurons (D1). Scale bar: 10 μm

FIG. 12: (A) Monoclonal antibody DC8E8 recognizes neurofibrillarydegeneration in transgenic rats SHR72. DC8E8 recognizes tau oligomericstage (arrows) and tangle stage (arrowhead) of tau neurodegeneration.Moreover, the antibody reacts with misfolded tau that is located in theaxonal fibers (inside the rectangle). (B) In age-matched control ratbrains the antibody does not display intraneuronal staining. Scale bar:20 μm. DC8E8 also recognizes all developmental stages of tangleformation in transgenic rat brain (SHR 72) as in human Alzheimer'sdisease. DC8E8 recognizes early developmental stages of tangleformation—monomeric, dimeric and early oligomeric stage (C) and lateoligomeric pre tangle stage (D), as well as late developmental stages ofpathological tau polymers—intracellular (E) and extracellularneurofibrillary tangles (missing nucleus) (F). Arrowhead in (C)indicates small oligomeric tau aggregates inside the neurons (A). Scalebar: 10 μm

FIG. 13: (A) DC8E8 staining of neurofibrillary tangles in the cortex ofSHR24 transgenic rats, which express tauΔ(1-150;392-441)/3R. (B) DC8E8recognized neurofibrillary tangles in the brainstem of the transgenicrats SHR72, which express tauΔ(1-150;392-441)/4R. Tissue sections werecounterstained with methylgreen. Arrows—neurofibrillary tangles. Scalebar: 50 μm

FIG. 14: Monoclonal antibody DC8E8 recognizes both soluble (A) andinsoluble tau protein (B) in the brain samples isolated from transgenicrat model SHR24 (isocortex) and Alzheimer's disease patients (allocortextissue including hippocampus, entorhinal and temporal cortex).Arrowhead—human truncated tau, arrow—rat endogenous tau. For soluble taufractions 15 μg of protein were loaded per lane. For insoluble taufractions the pellets were dissolved in 1× sodium dodecyl sulfate (SDS)samples loading buffer in 1/50 volume of the 1S, the same volume wereloaded as in the case of soluble fractions. Monoclonal antibody DC8E8recognizes both soluble and insoluble tau proteins in the brain samplesisolated from Alzheimer's disease patients (allocortex tissue includinghippocampus, entorhinal and temporal cortex) (C) and from transgenic ratmodel SHR72 (brain stem) (D). Arrow—physiological human tau proteins (A)and rat endogenous tau (B), arrowhead—human truncated tau(tauΔ(1-150;392-441)/4R) expressed as a transgene in the neurons ofSHR72 rats (D). For soluble tau fractions 15 μg of total protein wereloaded per lane. For insoluble tau fractions the pellets were dissolvedin 1× sodium dodecyl sulfate (SDS) samples loading buffer in 1/50 volumeof the 1S, the same volume were loaded as in the case of solublefractions

FIG. 15: DC8E8 inhibits pathological tau-tau interaction influorescence-based tau fibrillization assay. TauΔ(1-150;392-441)/4R(FIG. 15A) or tauΔ(1-296; 392-441)/4R (FIG. 15B) were induced by heparinto undergo a conformational change and fibrilize as measured byThioflavin T fluorescence; mAbs DC8E8, Rab50, and DC11 were tested fortheir ability to prevent the pathological conformation change.

FIG. 16: Analysis of the inhibitory potential of DC8E8 to prevent theformation of tau dimers, trimers, and oligomers by truncated tau proteintauΔ(1-296; 392-441)/4R by immunoblotting using HRP-conjugated mAb DC25.

FIG. 17: Uptake and degradation of TauΔ(1-150;392-441)/4R by microgliaBV2 cells. TauΔ(1-150;392-441)/4R was added to mouse BV2 cells eitheralone (1 μM) or in complex with monoclonal antibody DC8E8 (1 μMtauΔ(1-150;392-441)/4R+1 μM DC8E8). After incubation for various lengthsof time (2, 4, 6 and 12 hours), the BV2 cells were acid-washed, cellularproteins were extracted and the levels of internalized tau were analyzedby Western blotting with pan-tau antibody DC25. TauΔ(1-150;392-441)/4Rwas immunolabeled in cell lysates (intracellular tau) (A) and in cellcultivation medium (extracellular tau) (B). DC8E8 antibody wasvisualized with anti-mouse HRP-conjugated antibody. 20 μg of proteinwere loaded per lane.

FIG. 18: Stability (shelf-life) of DC8E8 at 37° C., as tested by ELISA.The antibody recognized tauΔ(1-150;392-441)/4R after several months ofstorage (1, 2, 3 and 4 months). The bars represent serial dilutions ofthe antibody as indicated. The measurements were performed intriplicate.

FIG. 19: DC8E8 recognizes and targets misfolded (diseased) tau in thebrain tissues of the human Alzheimer's disease. (A) Western blotanalysis with pan-tau DC25 antibody:

-   -   1) Biochemical extraction of pathological tau from the brain        tissues of human Alzheimer's disease (Greenberg and Davies,        1989);    -   2) Mock antibody (Rab50) does not recognize tau;    -   3) DC8E8 recognizes and targets misfolded (diseased) tau in        brain tissues of human Alzheimer's disease; and        (B) Ponceau S staining: 2), 3) Control of antibody amount (Rab50        and DC8E8) used in the experiment.

FIG. 20: DC8E8 recognizes and targets misfolded (diseased) tau in braintissues of the SHR72 rat model of AD. (A) Western blot analysis withpan-tau DC25 antibody:

-   -   4) Biochemical extraction of pathological tau from brain tissues        of human Alzheimer's disease (Greenberg and Davies, 1989);    -   5) Mock antibody (Rab50) does not recognize tau;    -   6) DC8E8 recognizes and targets misfolded (diseased) tau in        brain tissues of human Alzheimer's disease; and        (B) Ponceau S staining: 2), 3) Control of antibody amount (Rab50        and DC8E8) used in the experiment.

FIG. 21: In vivo, DC8E8 targets pathological forms of tau in the brainof transgenic rats (SHR72) and transports pathological tau from thebrain to the peripheral blood. (A) Concentration of the DC8E8 antibodyin the serum of DC8E8 treated animals reached 466, 200 and 273 μg/ml,respectively. (B) In vivo transport of the DC8E8-tau complexes from thebrain into the peripheral blood was observed. Pathological tau reachedthe average concentration of 350 μg/ml of the serum. Active transport oftau by DC8E8 eliminates pathological tau proteins from the brain. On theother hand, no tau proteins were detected in the sera of the animalstreated with mock antibody (Rab50), which recognizes the rabies virus(Macikova et al., 1992). Concentration of tau in the sera of the treatedanimals was determined by Innotest hTAU ELISA (Innogenetics, Belgium).The graph shows means with standard errors of the mean (SEM). Each ofthe 8 bars for rats A-C indicates a different sequential serum dilution(from 100-fold through 12,800-fold, from left to right).

FIG. 22: DC8E8 monoclonal antibody removes pathological tau from thebrain of transgenic rats (SHR72). (A) Intracerebral application of DC8E8(left panel) removes (arrows) pathological tau from the neurons incomparison with mock treated animals (right panel). (B) Quantificationof the amount of pathological tau in the neurons of the mock-treated andDC8E8-treated animals showed radical reduction in the amount ofpathological tau in animals treated with DC8E8 (p<0.0001).

FIG. 23: Recombinant scFv fragment (scDC8E8v) of monoclonal antibodyDC8E8, expressed in bacteria, recognizes pathological misdisorderedtauΔ(1-150;392-441)/4R. (A) Coomassie Brilliant Blue staining of crudelysates of control BL21 bacteria and bacteria harbouring scDC8E8vexpression plasmid, separated by 10% SDS-PAGE: lane 1, crude lysate ofcontrol BL21 bacteria; lane 2, crude lysate of BL21 bacteria expressingscDC8E8v; and lane M, protein molecular weight marker (Page RullerPrestained Protein Ladder #SM0672, Fermentas. (B) Ponceau S stainednitrocellulose membrane containing tau proteins: lane 1,tauΔ(1-150;392-441)/4R, 500 ng; lane 2, tauΔ(1-150;392-441)/4R, 250 ng;lane 3, tauΔ(1-150;392-441)/4R, 125 ng; lane 4 tauΔ228-441, 50 ng; andlane M, protein molecular weight marker. (C) Western blot/Nitrocellulosemembrane containing tau proteins, loaded as in B), detected with lysatefrom bacteria expressing scDC8E8v. (D) Western blot/Nitrocellulosemembrane containing tau proteins, loaded as in B), developed withnegative control bacterial lysate.

FIG. 24: Recombinant scFv fragment of monoclonal antibody DC8E8(scDC8E8v) exhibits tau binding properties similar to the DC8E8antibody-selectively recognizes tauΔ(1-150;392-441)/4R. (A) Kineticaffinity determination by SPR of scDC8E8v binding to ADtauΔ(1-150;392-441)/4R. (B) Kinetic affinity determination by SPR ofscDC8E8v binding to tau 2N4R. (C) Rate constants (k_(ON) and k_(OFF))and association equilibrium constant for scDC8E8v binding.

FIG. 25: Identification of residues in scDC8E8v combining site thatinfluence scDC8E8v/DC8E8's recognition of misdisordered tau. (A)Coomassie Brilliant Blue staining of polyacrylamide gels afterseparation of proteins from crude lysate of BL21 bacteria harboringscDC8E8v expression plasmid (wt) and its mutant forms. Each numberedlane corresponds to the respective clone number, (e.g. lane 2corresponds to 2-VL-R33A). Expressed single chain proteins are indicatedby asterisks. Control bacterial cultures do not express single chainproteins. (B) Ponceau S stained nitrocellulose membrane containing tauproteins: lane 1, tauΔ(1-150;392-441)/4R, 500 ng; lane 2,tauΔ(1-150;392-441)/4R, 250 ng; lane 3, tauΔ(1-150;392-441)/4R, 125 ng.(C) Western Blot on nitrocellulose membranes containing tau proteins,loaded as in B), detected with lysates from bacteria expressing eitherscDC8E8v (wt gel) or one of its mutant forms (blots 1-VL-N31A through22-VH-G102A).

FIG. 26: (A) Schematic of tau 2N4R (SEQ ID NO: 102) with the four DC8E8epitopes shown in hatched boxes within the enlarged region betweenresidues 261 and 373 (SEQ ID NO. 246): SEQ ID NOs 98-101, respectively.(1) Schematic of overlapping tau-derived peptide immunogens comprisingat least one of the four regions of tau recognized by the DC8E8antibody, for use as active vaccines or to purify DC8E8 antibodies andthe like; (2) general possibilities for other modified and designerpeptides and compounds, with optional moieties. (B) Summary ofimmunoblot analysis of insoluble tau prepared from the brain stems oftransgenic rats (SHR72, expressing tauΔ(1-150;392-441)/4R) treated withpeptides SEQ ID NOs 1-8 and 108. Immunoblot analysis was performed withvarious mAbs to determine the reduction of insoluble tau of thefollowing AD-relevant epitopes: mAb DC25 (tau 347-353), mAb DC217 (taupThr217), mAb DC209 (tau pThr 231), mAb AT8 (tau pSer202/pThr205) andmAb AT270 (tau pThr181). (C) Densitometric immunoblot analysis ofinsoluble tau prepared from the brain stems of rats treated with tau251-PDLKNVKSKIGSTENLKHQPGGGKVQIINK-280 (SEQ ID NO:1) combined withadjuvant and from control rats treated with adjuvant alone. Mean valuesare presented with standard error of the mean.

FIG. 27: Neurobehavioral evaluation of transgenic rats (SHR72) modelingAD treated with tau 251-PDLKNVKSKIGSTENLKHQPGGGKVQIINK-280 (SEQ IDNO:1). Ten days after 5^(th) dose of the immunogen, the transgenic ratswere used for behavioral testing. Diagrams represent mean±SEM. Allstatistical data were obtained using nonparametric Mann-Whitney U-test.(A) Beam walking test (3.5 beam). (B) Number of hindlimb slips (3.5beam). (C) Neuroscale.

FIG. 28: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:1 resulted in 49% reduction of neurofibrillary tangle (NFT) load.Antibody AT8 was used for evaluation of NFTs in the brain tissues oftransgenic rats SHR72.

FIG. 29: Quantitative immunoblot analysis of insoluble tau prepared fromthe brain stems of transgenic rats (SHR72) treated with tau256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285 (SEQ ID NO:2) with adjuvant orcontrol rats treated with adjuvant alone. Mean values are presented withstandard error of the mean.

FIG. 30: Neurobehavioral evaluation of transgenic rats (SHR72) treatedwith tau 256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285 (SEQ ID NO:2). Allstatistical data were obtained using nonparametric Mann-Whitney U-test.(A) Beam walking test (3.5 beam). (B) Number of hindlimb slips (3.5beam). (C) Neuroscale.

FIG. 31: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:2 resulted in 60% reduction of neurofibrillary tangle (NFT) load.Antibody AT8 was used for evaluation of NFTs in the brain tissues oftransgenic rats SHR72.

FIG. 32: Quantitative immunoblot analysis of insoluble tau prepared fromthe brain stems of transgenic rats (SHR72) treated with tau256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285 with phosphorylated Ser262 (SEQID NO:2) with adjuvant or control rats treated with adjuvant alone. Meanvalues are presented with standard error of the mean.

FIG. 33: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:2/Phospho resulted in 77% reduction of neurofibrillary tangle (NFT)load. Antibody AT8 was used for evaluation of NFTs in the brain tissuesof transgenic rats SHR72.

FIG. 34: Quantitative immunoblot analysis of insoluble tau prepared fromthe brain stems of transgenic rats (SHR72) treated with tau259-KIGSTENLKHQPGGGKVQIINKKLDLSNVQ-288 (SEQ ID NO:3) with adjuvant orcontrol rats treated with adjuvant alone. Mean values are presented withstandard error of the mean.

FIG. 35: Neurobehavioral evaluation of transgenic rats (SHR72) treatedwith tau 259-KIGSTENLKHQPGGGKVQIINKKLDLSNVQ-288 (SEQ ID NO:3). Allstatistical data were obtained using nonparametric Mann-Whitney U-test.(A) Beam walking test (3.5 beam). (B) Number of hindlimb slips (3.5beam). (C) Neuroscale.

FIG. 36: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:3 resulted in 58% reduction of neurofibrillary tangle (NFT) load.Antibody AT8 was used for evaluation of NFTs in the brain tissues oftransgenic rats SHR72.

FIG. 37: Quantitative immunoblot analysis of insoluble tau prepared fromthe brain stems of transgenic rats (SHR72) treated with tau275-VQIINKKLDL SNVQSKCGSKDNIKHVPGGG-304 (SEQ ID NO:4) or control ratstreated with adjuvant alone. Mean values are presented with standarderror of the mean.

FIG. 38. Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:4 showed moderate improvement in neurobehavioral parameters. (A) Beamwalking test (3.5 cm beam). (B) Number of hind-limb slips (3.5 cm beam).(C) Neuroscale. Data are presented as mean values with standard error ofthe mean.

FIG. 39: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:4 resulted in 66% reduction of neurofibrillary tangle (NFT) load.Antibody AT8 was used for evaluation of NFTs in the brain tissues oftransgenic rats SHR72.

FIG. 40: Immunoblot analysis of insoluble tau prepared from the brainstem of transgenic rats (SHR72) immunized with tau201-GSPGTPGSRSRTPSLPTPPT REPKKVAVVR-230/carrying phosphorylatedthreonine at position 217 (SEQ ID NO:5) with adjuvant or control ratstreated with adjuvant alone. Mean values are presented with standarderror of the mean.

FIG. 41: Neurobehavioral evaluation of transgenic rats (SHR72) treatedwith tau 201-GSPGTPGSRSRTPSLPTPPT REPKKVAVVR-230/carrying phosphorylatedthreonine at position 217 (SEQ ID NO:5). All statistical data wereobtained using nonparametric Mann-Whitney U-test. (A) Beam walking test(3.5 beam). (B) Number of hindlimb slips (3.5 beam). (C) Neuroscale.

FIG. 42: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:5 showed no effect on neurofibrillary tangle (NFT) load. Antibody AT8was used for evaluation of NFTs in the brain tissues of transgenic ratsSHR72.

FIG. 43: Immunoblot analysis of insoluble tau prepared from the brainstem of transgenic rats (SHR72) immunized with tau379-RENAKAKTDHGAEIVYKSPVVSGDTSPRHL-408 carrying phosphorylated serineresidues at position 396 and 404 (SEQ ID NO:6) and adjuvant or controlrats treated with adjuvant alone. Mean values are presented withstandard error of the mean.

FIG. 44: Neurobehavioral evaluation of transgenic rats (SHR72) treatedwith tau SEQ ID NO:6 phosphorylated at Ser396/Ser404. All statisticaldata were obtained using nonparametric Mann-Whitney U-test. (A) Beamwalking test (3.5 beam). (B) Number of hindlimb slips (3.5 beam). (C)Neuroscale.

FIG. 45: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:6 showed no reduction of neurofibrillary tangle (NFT) load. AntibodyAT8 was used for evaluation of NFTs in the brain tissues of transgenicrats SHR72.

FIG. 46: Immunoblot analysis of insoluble tau prepared from the brainstem of rats(SHR72) immunized with tau181-TPPSSGEPPKSGDRSGYSSPGSPGTPGSRS-210 carrying phosphorylated serineresidue at position 202 and threonine residue at 205 (SEQ ID NO:7) withadjuvant or control rats treated with adjuvant alone. Mean values arepresented with standard error of the mean.

FIG. 47: Neurobehavioral evaluation of SHR72 rats treated with tau181-TPPSSGEPPKSGDRSGYSSPGSPGTPGSRS-210 carrying phosphorylated serineresidue at position 202 and threonine residue at 205 (SEQ ID NO:7). Allstatistical data were obtained using nonparametric Mann-Whitney U-test.(A) Beam walking test (3.5 beam). (B) Number of hindlimb slips (3.5beam). (C) Neuroscale.

FIG. 48: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:7 showed no effect on neurofibrillary tangle (NFT) load. Antibody AT8was used for evaluation of NFTs in the brain tissues of transgenic ratsSHR72.

FIG. 49: Immunoblot analysis of insoluble tau prepared from the brainstem of rats (SHR72) immunized with tau 300-VPGGGSVQIVYKPVDLSK-317 (SEQID NO:8) with adjuvant or control rats treated with adjuvant alone. Meanvalues are presented with standard error of the mean.

FIG. 50: Neurobehavioral evaluation of transgenic AD rats (SHR72)treated with tau 300-VPGGGSVQIVYKPVDLSK-317 (SEQ ID NO:8). Allstatistical data were obtained using nonparametric Mann-Whitney U-test.(A) Beam walking test (3.5 beam). (B) Number of hindlimb slips (3.5beam). (C) Neuroscale.

FIG. 51: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:8 showed no reduction of neurofibrillary tangle (NFT) load. AntibodyAT8 was used for evaluation of NFTs in the brain tissues of transgenicrats SHR72.

FIG. 52: Vaccination of transgenic rats SHR72 with tau peptide (SEQ IDNO:108) statistically significantly reduced insoluble pathological tau(p<0.001). Pathological insoluble tau was extracted from the brains oftransgenic rats SHR72 immunized with tau peptide and analyzed byimmunoblotting. Mean values are presented with standard error of themean.

FIG. 53: Vaccination of transgenic rats SHR72 with tau peptide (SEQ IDNO:108) statistically significantly improved neurobehavioral parameters(p<0.05). (A) Beam walking test (3.5 cm beam). (B) Number of hind-limbslips (3.5 cm beam). (C) Neuroscale. Data are presented as mean valueswith standard error of the mean.

FIG. 54: Vaccination of transgenic rats SHR72 with tau peptide (SEQ IDNO:108) resulted in 60% reduction of neurofibrillary tangle (NFT) load.Antibody AT8 was used for evaluation of NFTs in the brain tissues oftransgenic rats SHR72.

FIG. 55: ELISA of antisera generated from immunization of transgenicrats (SHR72) with peptide tau 275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304(SEQ ID NO:4) shows a difference in the antisera's binding to humanpathological tauΔ(1-150; 392-441)/4R and human physiological tau 2N4R.

FIG. 56: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:108 induced formation of antibodies preferentially binding topathological tau protein. Geometric mean antibody titers measured withELISA show that antibodies elicited by vaccination with tau peptide SEQID NO:108 exhibited highest binding activity to immunogen (SEQ ID NO:108peptide) and to pathological tauΔ(1-150; 392-441)/4R. Physiological tau(tau2N4R), which was used as a control, was more weakly recognized.

FIG. 57: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:108 preferentially induced formation of IgG antibody isotypesspecific to pathological tau. The isotype profile of antibodies inducedby tau peptide SEQ ID NO:108 is shown. Sera from individual rats werediluted 1:800 and binding activity to pathological tauΔ(1-150;392-441)/4R was analyzed by ELISA.

FIG. 58: SPR affinity determination of antisera generated fromimmunization of SHR72 rats with peptide tau275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304 (SEQ ID NO:4) for binding tohuman tauΔ(1-150;392-441)/4R and human tau 2N4R.

FIG. 59: Immunohistochemical staining of brains from a human AD patientwith rat antibodies generated from immunization of transgenic rats(SHR72) with tau 275-VQIINKKLDL SNVQSKCGSKDNIKHVPGGG-304 (SEQ ID NO: 4).(A) The antisera recognized neurofibrillary lesions in Alzheimer'sdisease brain, hippocampus. (B) Higher magnification showedneurofibrillary tangles. Scale bars: 100 μm (A), 10 μm (B).

FIG. 60: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:108 induced antibodies recognizing pathological tau proteins insections from human Alzheimer's disease brain tissues. Representativeimmunostaining of the rat serum Nos. 3 (A), 5 (B), 6 (C), 7 (D), and 8(E) show that all tested rat serum antibodies recognized neurofibrillarytangles in Pre-α layer of the entorhinal cortex of an Alzheimer'sdisease patient. Pooled sera from rats immunized with adjuvant only wereused as a negative control (F). Serial brain tissue sections from theentorhinal cortex were used. Scale bar: 50 μm.

FIG. 61: Vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:108 induced specific antibodies recognizing pathological tau proteinsin human Alzheimer's disease brains as well as in the brains oftransgenic rats SHR72. Pathological tau was extracted from human and ratbrain tissues and analyzed by immunoblotting with pooled sera frompeptide SEQ ID NO:108 immunized transgenic rats SHR72. The seraantibodies recognized monomeric (lane No. 1, 2 and No. 3) and oligomeric(lane No. 2 and No. 3) pathological tau including AD characteristic A68pathological tau.

FIG. 62: Immunization of mice with tau peptide SEQ ID NO:109 inducedantibodies with statistically significantly higher binding activity topathological tauΔ(1-150; 392-441)/4R than to physiological tau 2N4R(p=0.0115). The graph represents statistical evaluation of ELISA resultsfor individual sera diluted at 1:800. Mean values are shown withstandard error of the mean.

FIG. 63: Immunization of mice with tau peptide SEQ ID NO:110 inducedantibodies exhibiting statistically significantly higher bindingactivity to pathological tauΔ(1-150; 392-441)/4R than to physiologicaltau 2N4R (p=0.0029). The graph represents statistical evaluation ofELISA results for individual sera diluted at 1:800. Mean values areshown with standard error of the mean.

FIG. 64: Immunization of mice with tau peptide SEQ ID NO:111 inducedantibodies exhibiting statistically significantly higher bindingactivity to pathological tauΔ(1-150; 392-441)/4R than to physiologicaltau 2N4R (p=0.0007). The graph represents statistical evaluation ofELISA results for individual sera diluted at 1:800. Mean values areshown with standard error of the mean.

FIG. 65: Immunization of mice with tau peptide SEQ ID NO:112 inducedantibodies exhibiting statistically significantly higher bindingactivity to pathological tauΔ(1-150; 392-441)/4R than to physiologicaltau 2N4R (p<0.001). The graph represents statistical evaluation of ELISAresults for individual sera diluted at 1:800. Mean values are shown withstandard error of the mean.

FIG. 66: Designer therapeutic epitopes GWSIHSPGGGSC (SEQ ID NO: 250) andSVFQHLPGGGSC (SEQ ID NO: 251) competed with pathologicaltauΔ(1-150;392-441)/4R for binding to antibody DC8E8.

FIG. 67: Designer therapeutic epitopes GWSIHSPGGGSC (SEQ ID NO: 250) andSVFQHLPGGGSC (SEQ ID NO: 251) induced production of antibodies thatstatistically significantly discriminated between pathologicaltauΔ(1-150;392-441)4R and physiological tau 2N4R, as assayed by ELISA.Sera (at 1:3200 dilution) from mice immunized with one of the peptides250 and 251 were tested for antibodies specific for tau proteins:pathological tauΔ(1-150;392-441)4R and physiological tau 2N4R by ELISA.

FIG. 68: Immunization with designer therapeutic epitopes GWSIHSPGGGSC(SEQ ID NO: 250) and SVFQHLPGGGSC (SEQ ID NO: 251) induced the mostrobust production of IgG1-isotype antibodies.

FIG. 69: Quantitative SPR (Surface Plasmon Resonance) measurements showthat antibodies induced by designer therapeutic epitope 1 (GWSIHSPGGGSC,SEQ ID NO: 250) (FIG. 69A) and designer therapeutic epitope 2(SVFQHLPGGGSC, SEQ ID NO: 251) (FIG. 69B) statistically significantly(p<0.001 and p<0.01, respectively) discriminated between pathologicaltauΔ(1-150;392-441)/4R and physiological 2N4R tau.

FIG. 70: Immunohistochemical staining of human AD diseased brain tissueswith sera generated against designer therapeutic epitope 1(GWSIHSPGGGSC, SEQ ID NO: 250) and designer therapeutic epitope 2(SVFQHLPGGGSC, SEQ ID NO: 251). (A) Antisera against designertherapeutic epitope 1 recognized neurofibrillary pathology in the brainof AD patient. (C) High magnification of the neurofibrillary tangle andneuropil threads (arrows). (B) Antisera against designer therapeuticepitope 2 recognized neurofibrillary pathology in the brain of ADpatient. (D) High magnification of the stained neurofibrillary tangleand neuropil threads (arrows). Antisera against designer therapeuticepitope 1 and designer therapeutic epitope 2 did not recognize normaltau in the control human brain (E, F), scale bar: 50 μm (A, B, E, F), 20μm (C, D). (G) Serum generated against designer therapeutic epitope 2(SVFQHLPGGGSC, SEQ ID NO: 251) recognizes neurofibrillary lesions intransgenic rats SHR72. (H) In age-matched control rat brains theantibody does not display intraneuronal staining. The serum recognizesoligomeric pre tangle stage (I), as well as intracellular (J). Scalebar: 20 μm (A, B), 10 μm (C, D).

FIG. 71: Antibodies induced by designer therapeutic epitope 1(GWSIHSPGGGSC, SEQ ID NO: 250) and designer therapeutic epitope 2(SVFQHLPGGGSC, SEQ ID NO: 251) recognized soluble and sarkosyl-insolublepathological tau isolated from the human Alzheimer's disease braintissues.

FIG. 72: Antibodies induced by designer therapeutic epitope 1(GWSIHSPGGGSC, SEQ ID NO: 250) and designer therapeutic epitope 2(SVFQHLPGGGSC, SEQ ID NO: 251) recognized soluble (Lane 1, 3, 5) andinsoluble (Lane 2, 4, 6) pathological tau isolated from the brains ofthe Alzheimer's disease rat model (SHR72).

FIG. 73: Immunotherapy with designer therapeutic epitope 2(SVFQHLPGGGSC, SEQ ID NO: 251) showed significant improvement inneurobehavioral parameters (Neuroscale) of treated SHR72 rats. (A) Beamwalking test. (B) Number of hind-limb slips (p<0.05). (C) Neuroscale.Rats treated with the designer therapeutic epitope 2 (SEQ ID NO: 251)showed: a) decreased escape latencies by 27% in the beam walking test,b) reduced number of the hind-limb slips by 44% (p<0.05), and c) thereduced Neuroscale score by 26% than the transgenic control rats thatreceived adjuvant alone. All statistical data were obtained usingnonparametric Mann-Whitney U-test.

FIG. 74: Immunotherapy with designer therapeutic epitope 2(SVFQHLPGGGSC, SEQ ID NO: 251) showed a statistically significantreduction of pathological tau in brains of immunized Alzheimertransgenic SHR72 rats. Immunotherapy statistically significantly(p<0.05) reduced the amount of pathological insoluble tau in immunizedanimals compared to the control transgenic rats that received adjuvantalone. The reduction of pathological insoluble tau was observed at allanalyzed tau epitopes (P<0.05).

FIG. 75: (A) Schematic of synthetic peptides used for further evaluationof DC8E8's minimal epitope (therapeutic core unit) and immunogenicpotency determination and (B) their amino acid sequences.

FIG. 76: Determination of DC8E8 minimal epitope (therapeutic core unit)using synthetic peptides by competitive ELISA. Ten tau peptides (SEQ IDNOs: 270, 271, 272, 275, 276, 277, 280, 281, 282 and 283) that containat least 6 amino acids of the DC8E8 recognition sequence are capable tocompete with pathological tauΔ(1-150;392-441)/4R for binding to antibodyDC8E8. Tau peptides containing only 5 amino acids of the DC8E8recognition sequence (SEQ ID NOs: 273, 274, 278 and 279) do not competewith tauΔ(1-150;392-441)/4R (SEQ ID NO: 199) for binding to antibodyDC8E8.

FIG. 77: Induction of tau specific antibodies after immunization ofC57BL mice with tau peptides. (A) 12-mer, 7-mer and 6-mer peptides (SEQID NOs: 270, 271 and 272, respectively) are immunogenic. The antibodiesinduced by immunization exhibit statistically significantly higherbinding activity to pathological tauΔ(1-150; 392-441)/4R than tophysiological tau 2N4R (p<0.0079; p<0.0052; p<0.0079, respectively).5-mer peptides SEQ ID NOs: 273 and 274 are not immunogenic. (B) 42-mer,19-mer, 7-mer and 6-mer peptides (SEQ ID NOs: 275, 280, 276 and 277,respectively) are immunogenic. Antibodies induced by these peptidesstatistically significantly (p<0.0079, p<0.0159, p<0.0079 and p<0.0379,respectively) discriminated between pathological tauΔ(1-150;392-441)/4Rand physiological tau 2N4R. 5-mer peptides SEQ ID NOs: 278 and 279 arenot immunogenic. (C) 7-mer peptides (SEQ ID NOs: 281 and 283) areimmunogenic. Antisera against these peptides statistically significantly(p<0.0379, and p<0.0286, respectively) discriminated betweenpathological tauΔ(1-150;392-441)/4R and physiological tau 2N4R. Thelevels of antibodies to pathological tau and physiological tau inducedby 6-mer peptide SEQ ID NO: 282 were very low. The graphs representstatistical evaluation of ELISA results for individual sera diluted at1:800. Mean values are shown with standard error of the mean.

FIG. 78: Geometric mean antibody titers of tau specific antibodies afterimmunization of C57BL mice with tau peptides. Vaccination of C57BL micewith tau peptide SEQ ID NOs: 270, 271, 272, 275, 276, 277, 280, 281 and283 induced formation of tau specific antibodies. Geometric meanantibody titers measured with ELISA show that antibodies elicited byvaccination with tau peptide SEQ ID NOs: 270, 271, 272, 275, 276, 277,280, 281 and 283 exhibited higher binding activity to pathologicaltauΔ(1-150; 392-441)/4R than to physiological tau (tau2N4R). Lowertiters of tau specific antibodies were detected after immunization ofmice with tau peptides SEQ ID NOs: 273, 274, 278, 279 and 282.

FIGS. 79A and 79B: The isotype profile of antibodies induced by taupeptides is shown. Immunization of C57/BL mice with tau peptidescarrying minimal DC8E8 epitope preferentially induced formation of IgG1and IgG2b antibody isotypes specific to pathological tau. Pooled serafrom individual mice were diluted 1:800 and binding activity topathological tauΔ(1-150; 392-441)/4R was analyzed by ELISA.

FIG. 80: Quantitative evaluation of the binding capacity of antibodies,which were induced in mice C75BL immunized with tau peptides, totauΔ(1-150;392-441)/4R and 2N4R. Surface plasmon resonance (SPR)measurements showed that antibodies against tau peptides SEQ ID NOs:270, 271, 272, 275, 276, 277, 280, 281 and 283 statisticallysignificantly (** . . . p<0.001 and * . . . p<0.01) discriminatedbetween pathological tauΔ(1-150;392-441)/4R and physiological 2N4R tau.KA—the association equilibrium binding constant.

FIG. 81: Antibodies induced in mice immunized with tau peptidesrecognize pathological forms of tau in Western blotting. Vaccination ofC57BL mice with tau peptides SEQ ID NOs: 270, 271, 272, 275, 276, 277,280, 281 and 283 induced specific antibodies, which recognizepathological tau proteins isolated from human Alzheimer's disease braintissue as well as from the brain stems of transgenic rats SHR72.Antisera after immunization of mice with peptides SEQ ID NOs: 273, 274,278, 279 and 282 did not recognize pathological tau forms.

FIGS. 82A-C: Neurofibrillary tangles recognized by tau peptide-inducedantibodies in human AD brain tissues. Vaccination of C57BL mice with taupeptides SEQ ID NOs: 270, 271, 272, 275, 276, 277, 280, 281 and 283induced antibodies recognizing neurofibrillary lesions in hippocampus ofAlzheimer's disease brain. Sera from mice immunized with adjuvant onlywere used as a negative control. Brain tissue sections from thehippocampus CA1 were used. Scale bar: 100 μm.

FIG. 83: Summary of immunohistochemical staining (and respectiverelative intensities) of brain tissues from a human AD patient with seraantibodies generated from immunization of C57BL mice with tau peptidesSEQ ID NOS: 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, and 283.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibody” refers to an immunoglobulin, whether geneticallyengineered, natural, or wholly or partially synthetically orrecombinantly produced. All derivatives, portions, and fragments thereofthat maintain antigen-binding properties and at least one of thetau-related characteristic properties according to the invention arealso included in the term. The term also encompasses any protein havinga binding domain which is homologous or largely homologous to animmunoglobulin binding domain. These proteins can be derived fromnatural sources, or partly or wholly synthetically or recombinantlyproduced. An antibody can be monoclonal or polyclonal. The antibody canbe a member of any immunoglobulin class, including any of the humanclasses: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class arepreferred in some embodiments of the present invention.

The terms “isolated antibody” and “isolated peptide” refer to a proteinor peptide produced from cDNA-, recombinant RNA-, or any othersynthetic-origin, or some combination thereof; as well as to proteinsand peptides that, by virtue of their origin, or source of derivation,either (1) are not associated with proteins found in nature, (2) arefree of other proteins from the same source, e.g. free of murineproteins, (3) are expressed by a cell from a different species, or (4)do not occur in nature.

The antibodies according to the invention include, in addition, suchantibodies having “conservative sequence modifications,” nucleotide andamino acid sequence modifications which do not affect or alter theabove-mentioned characteristics of the antibody according to theinvention. Modifications can be introduced by standard techniques knownin the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a anti-tau antibody can befor example replaced with another amino acid residue from the same sidechain family.

“Antibody fragments” and “antibody portions” comprise a portion of afull length antibody, generally at least the antigen bindingportion/domain or the variable region thereof. Examples of antibodyfragments include diabodies, single-chain antibody molecules,immunotoxins, and multispecific antibodies formed from antibodyfragments. In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a VH chain bindingpathological tau, namely being able to assemble together with a VL chainor of a VL chain binding to pathological tau, namely being able toassemble together with a VH chain to form a functional antigen bindingpocket and thereby providing the property of binding to pathologicaltau. The terms also comprise fragments that per se are not able toprovide effector functions (e.g., ADCC/CDC) but provide this functionafter being combined with the appropriate antibody constant domain(s).

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are especially preferred. Such murine/human chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding murine immunoglobulin variable regions and DNAsegments encoding human immunoglobulin constant regions. Other forms of“chimeric antibodies” encompassed by the present invention are those inwhich the class or subclass has been modified or changed from that ofthe original antibody. Such “chimeric” antibodies are also referred toas “class-switched antibodies.” Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques now known in the art. See, e.g., Morrison, S. L., et al.,Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos. 5,202,238and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework regions (FR) and/or the complementarity determining regions(CDR) have been modified to comprise the CDR of an immunoglobulin ofdifferent specificity as compared to that of the parent immunoglobulin.In one embodiment, a murine CDR is grafted into the framework region ofa human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigens andepitopes described herein as “therapeutic epitopes” on tau.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The constant regions of the antibodycan be, for example, constant regions of human IgG1 type. Such regionscan be allotypic and are described by, e.g., Johnson, G., and Wu, T. T.,Nucleic Acids Res. 28 (2000) 214-218 and the databases referencedtherein, and are preferentially useful for some embodiments, as long asthe properties of induction of ADCC and for example CDC according to theinvention are retained.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NSO or CHO cell or from an animal (e.g. a mouse such as aXENOMOUSE, a genetically modified mouse that produces antibodies havingamino acid sequences of human antibodies, e.g., human framework (FR) andhuman constant region amino acid sequences) that is transgenic for humanimmunoglobulin genes or antibodies expressed using a recombinantexpression vector transfected into a host cell. Such recombinant humanantibodies have variable and constant regions derived from humangermline immunoglobulin sequences in a rearranged form. The recombinanthuman antibodies according to the invention have been subjected to invivo somatic hypermutation. Thus, the amino acid sequences of the VH andVL regions of the recombinant antibodies are sequences that, whilederived from and related to human germline VH and VL sequences, can notnaturally exist within the human antibody germline repertoire in vivo.

The term “effector functions” includes, but is not limited to, C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; anddown regulation of cell surface receptors (e.g. B cell receptor; BCR).

The term “epitope” is used here to refer to binding sites recognized bya binding protein or an antibody. Epitopes can be any molecule orgrouping thereof, including, but not limited to, amino acids, amino acidside chains, sugars, and lipids, and can have a specificthree-dimensional structure or conformation. Thus, an epitope cancomprise any portion of a tau peptide/protein molecule that includesprimary, secondary, tertiary, or quaternary structure, as those termsare generally known in the art. A “linear epitope” is made up of acontinuous sequence of amino acid residues. A linear epitope is one thatis present on physiological tau (e.g., is present in tau 2N/4R). A“conformational epitope” is an epitope to which the antibody or bindingprotein binds in a conformational-specific manner. In the case ofprotein-based epitopes, the binding can depend on theepitope-carrying-protein's secondary, tertiary, or quaternary structure.In other words, the antibody binds in a structure-specific manner, atertiary-structure-specific manner, or a quaternary-structure-specificmanner. A conformational epitope is one that is present in pathologicaltau (e.g., present in tauΔ(1-150;392-441)/4R)).

The term “therapeutic epitope” refers to regions within tau that wereidentified herein and were found to promote tau-tau aggregation, when incertain conformations (recognized by the DC8E8 antibody). Antibodies(and other binding proteins) that bind to one or more of these regionsinhibit early and late stages of tau aggregation, including theconversion of tau monomer to dimer, and conversion to higher aggregateforms; i.e., the antibodies inhibit the conversion from physiologicaltau to pathological tau. These regions within tau can be involved inpromoting tau fibrillization into paired helical filaments (PHFs), bypromoting the formation of beta-sheets within adjacent regions of tau.Therapeutic epitopes are comprised within 267-KHQPGGG-273 (within 1^(st)repeat domain of tau protein), 298-KHVPGGG-304 (within 2^(nd) repeatdomain of tau protein), 329-HHKPGGG-335 (within 3^(rd) repeat domain oftau protein), and 361-THVPGGG-367 (within 4^(th) repeat domain of tauprotein. In some embodiments, the therapeutic epitopes are eachcomprised within 268-HQPGGG-273 (within 1^(st) repeat domain of tauprotein), 299-HVPGGG-304 (within 2^(nd) repeat domain of tau protein),330-HKPGGG-335 (within 3^(rd) repeat domain of tau protein), and362-HVPGGG-367, respectively.

The term “displaying a higher affinity for pathological tau than forphysiological tau” refers to a higher degree of interaction between theantibody and at least one form of pathological tau than between theantibody and at least one form of physiological tau. The interaction canbe measured by, e.g., ELISA or surface plasmon resonance (SPR), asdescribed in the EXAMPLES below.

The terms “specifically binds,” “binds specifically,” and “specific to,”are interchangeable and mean that an antibody or antigen-bindingfragment thereof (or other binding protein) forms a complex with anantigen or epitope that is relatively stable under physiologicconditions. Specific binding can be characterized by a dissociationconstant of about 1×10⁻⁶ M or smaller, for example less than about 100nM, and most for example less than 10 nM. Methods for determiningwhether two molecules specifically bind are known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. Typically, an antibody or antigen-binding fragment thereofprovided by the invention is a molecule that binds the antigen or anepitope with such a dissociation constant of at least about 1×10⁻⁶ M orsmaller, but does not bind other molecules with such a dissociationconstant.

“Preferentially bind” refers to binding with higher affinity topathological tau than to physiological tau, for example, binding withhigher affinity to tauΔ(1-150;392-441)/4R than to 2N4R.

A “universal T-cell epitope” is a sequence selected from InfluenzaHemagluttinin: HA307-319 (PKYVKQNTLKLAT) (SEQ ID NO: 123); PADRE(AKXVAAWTLKAAA) (SEQ ID NO: 124); Malaria CS: T3 epitope(EKKIAKMEKASSVFNV) (SEQ ID NO: 125); Hepatitis B surface antigen:HBsA919_(—)28 (FFLLTRILTI) (SEQ ID NO: 126); Heat Shock Protein 65:hsp65153_(—)171 (DQSIGDLIAEAMDKVGNEG) (SEQ ID NO: 127); bacilleCalmette-Guerin (QVHFQPLPPAWKL) (SEQ ID NO: 128); Tetanus toxoid:TI830-844 (QYIKANSKFIGITEL) (SEQ ID NO: 129); Tetanus toxoid: TI947-967(FNNFTVSFWLRVPKVSASHLE) (SEQ ID NO: 130); and HIV gp120 T1(KQIINMWQEVGKAMYA) (SEQ ID NO: 131).

The term “intrinsically disordered tau” refers to thenormal/physiological form of tau protein, which lacks any defined 3Dstructure. It exists in the healthy brain (Kovacech et al., 2010).

“Misdisordered tau” refers to the forms of tau that differconformationally from normal/physiological intrinsically disordered tau,and does not have a firm/defined 3D-structure. Misdisordered truncatedtau is able to induce neurofibrillary degeneration in vivo. It does notexist in a healthy brain (Kovacech et al., 2010). “Misordered tau”refers to a structured pathological form of tau assembled into polymersof PHFs, which form NFTs. Misordered tau does not exist in a healthybrain (Kovacech et al., 2010).

“SHR24” refers to transgenic rat line that expresses tau type IIB(151-391/R3). The transgenic rats developed progressive age-dependentneurofibrillary degeneration in the cortical brain areas.Neurofibrillary tangles (NFTs) in SHR24 rats satisfy several keyhistological criteria used to identify neurofibrillary degeneration inhuman Alzheimer's disease including argyrophilia, Congo redbirefringence, and Thioflavin S reactivity. These criteria can be usedfor analysis of neurofibrillary degeneration in subjects receiving anyof the embodiments of the invention. Neurofibrillary tangles were alsoidentified with antibodies used to detect pathologic tau in the humanbrain, including DC11, recognizing an abnormal tau conformation andantibodies that are specific for hyperphosphorylated forms of tauprotein. Moreover, neurofibrillary degeneration was characterized byextensive formation of sarkosyl insoluble tau protein complexesconsisting of rat endogenous and truncated tau species (Filipcik et al.,2010).

“SHR72” refers to transgenic rats that express human truncatedtauΔ(1-150;392-441)/4R according to the International Patent ApplicationPCT WO 2004/007547), in several brain regions and spinal cord.Generation of this rat line was described by Zilka et al., 2006, and taupathology was described in Koson et al., 2008.

“Tau type IA” refers to N- and C-terminally double truncated tauproteins that have at least the first 236 N-terminal amino acids and atleast the last 45 C-terminal amino acids of the 4 repeat containingtau43 truncated. The molecules are detectable in Alzheimer's diseasedbrain tissue whereas the molecules are not detectable in normal healthybrain tissue (WO2004/007547 A2).

“Tau type IB” refers to N- and C-terminally double truncated tauproteins that have at least the first 236 N-terminal amino acids and atleast the last 45 C-terminal amino acids of the 3 repeat containingtau44 truncated. The molecules are detectable in Alzheimer's diseasedbrain tissue whereas the molecules are not detectable in normal healthybrain tissue (WO2004/007547 A2).

“Tau type IIA” refers to N- and C-terminally double truncated tauproteins that have at least the first 68 N-terminal amino acids and atleast the last 40 C-terminal amino acids of the 4 repeat containingtau43 truncated. The molecules are detectable in Alzheimer's diseasedbrain tissue whereas the molecules are not detectable in normal healthybrain tissue (WO2004/007547 A2).

“Tau type IIB” refers to N- and C-terminally double truncated tauproteins that have at least the first 68 N-terminal amino acids and atleast the last 20 C-terminal amino acids of the 3 repeat containingtau44 truncated. The molecules are detectable in Alzheimer's diseasedbrain tissue whereas the molecules are not detectable in normal healthybrain tissue (WO2004/007547 A2).

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect can be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or can be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, also coversany treatment of AD or related tauopathies in a mammal, particularly ina human, and includes: (a) preventing the disease from occurring in asubject which can be predisposed to the disease or at risk of acquiringthe disease but has not yet been diagnosed as having it; (b) inhibitingthe disease, i.e., arresting its development; and (c) relieving thedisease, i.e., causing regression of the disease. Preferred embodimentsof “treatment” are further discussed below. In some embodiments,“treating” refers to administering a therapeutic agent to a patientsuspected of suffering or already suffering from AD or anothertauopathy. It can also refer to reducing, eliminating, or at leastpartially arresting, as well as to exerting any beneficial effect, onone or more symptoms of the disease and/or associated with the diseaseand/or its complications.

“Prevention” refers to administration to a patient susceptible to, orotherwise at risk of, a particular disease. Anyone in the generalpopulation is at risk for AD. Some individuals have an increased,genetic risk for AD. Prevention can eliminate or reduce the risk ordelay the onset of disease. Delay of onset or progression can bemeasured based on standard times of disease progression in similarpopulations or individuals.

“Tauopathy” refers to a disease associated with the formation ofpathological tau.

“Physiological tau” refers to any one of the 6 isoforms of normal humantau, namely:

2N4R (SEQ ID NO: 102) 1N4R (SEQ ID NO: 103) 2N3R (SEQ ID NO: 104) 0N4R(SEQ ID NO: 105) 1N3R (SEQ ID NO: 106) 0N3R (SEQ ID NO: 107)

Excluded from this definition are those that carry any one of thephosphorylations associated with Alzheimer's disease and othertauopathies.

“Pathological tau” includes pathological tau conformers and structuresand encompasses all of the following: Tau Type IA, IB, IIA, and IIB,misordered, misdisordered tau (monomer, dimer, trimer, oligomer),misdisordered soluble tau, sarkosyl-insoluble tau, extracellular taudeposits, tau aggregates, paired helical filaments, neurofibrillarypathology, including neurofibrillary lesions, tangles, threads, fibrils,axonal spheriods, highly phosphorylated forms of truncated tau and offull-length tau, or any other form of tau associated with AD or anothertauopathy.

“Linked” refers to attachment of a moiety to a peptide, antibody, orcompound. The moiety can be coupled, or complexed, or covalently ornon-covalently attached. The moiety can be chemically crosslinked orexpressed or synthesized as a fusion with the peptide or antibody.

“Moiety” refers to any compound, organic, peptide, protein, nucleicacid, carrier, adjuvant, that is able to be attached to the peptide,antibody, or binding protein, but that is not the claimed peptide,antibody, or binding protein itself.

“Immunogenic” refers to something that can elicit an immune response.The immune response can be antibody- or cell-mediated, or both.

“Adjuvant” refers to a substance that is capable of increasing,amplifying, or modulating the immune response to the accompanyingpeptide.

“Other therapy” refers to additional therapies that the subject patientscan receive.

“Clearance” refers to a reduction in levels or detection of pathologicaltau and/or a pathological tau structure. Clearance does not have to be acomplete disappearance of pathological tau, i.e., it can be a partialdisappearance.

The term “promoting” encompasses inducing, improving, or increasing.

“Brain tissue” refers to any neuronal tissue, e.g., from the brain,brain stem, and spinal cord.

The term “specific binding” and “high affinity”, respectively, refers toantibody binding to a predetermined antigen, i.e. the tau epitopedefined above. Typically, the antibody binds with a dissociationconstant (KD) of 10⁻⁶ M or less, and binds to the predetermined antigenwith a KD that is at least twofold less than its KD for binding to anonspecific antigen (e.g., BSA, casein, or any other specifiedpolypeptide) other than the predetermined antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”. As used herein “highlyspecific” binding means that the relative K_(D) of the antibody formisdisordered tau is at least 4-fold less than the K_(D) for bindingthat antibody to other ligands or to normal full-length tau.

The term “prokaryotic” is meant to include all bacteria which can betransformed or transfected with a DNA or RNA molecule for the expressionof an antibody of the invention or one or more of the correspondingimmunoglobulin chains. Prokaryotic hosts can include gram negative aswell as gram positive bacteria such as, for example, E. coli, S.typhimurium, Serratia marcescens and Bacillus subtilis. The term“eukaryotic” is meant to include yeast, higher plant, insect and forexample mammalian cells, most for example HEK 293, NSO, and CHO cells.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties that are not normally a part of the basemolecule. Such moieties can improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties canattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule.

The terms “nucleic acid”, “nucleic sequence”, “nucleic acid sequence”,“polynucleotide,” “oligonucleotide”, “polynucleotide sequence” and“nucleotide sequence” are used interchangeably in the presentdescription, and refer to a precise sequence of nucleotides, modified ornot, defining a fragment or a region of a nucleic acid, containingunnatural nucleotides or not, and being either a double-strand DNA, asingle-strand DNA or transcription products of said DNAs.

The term “isolated polynucleotide” or “isolated nucleic acid” as usedherein shall mean a polynucleotide of genomic, cDNA, or synthetic originor some combination thereof, which by virtue of its origin either (1) isnot associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

Antibodies for Diagnostics, Passive Immunization, Drug Delivery, andAD-Therapy

Described herein are novel isolated antibodies, specific to one or moretau epitopes displayed by pathological forms of tau. These epitopes arelocated within regions of tau that are for the first time assigned arole in pathological tau aggregation, namely within: 267-KHQPGGG-273(SEQ ID NO: 98) (i.e., epitope #1 is located within 267-KHQPGGG-273,which falls within thei^(st) repeat domain of tau protein),298-KHVPGGG-304 (SEQ ID NO: 99) (epitope #2, within the 2^(nd) repeatdomain of tau protein), 329-HHKPGGG-335 (SEQ ID NO: 100) (epitope #3,within the 3^(rd) repeat domain of tau protein), and 361-THVPGGG-367(SEQ ID NO: 101) (epitope #4, within the 4^(th) repeat domain of tauprotein). These antibodies are capable of recognizing misordered andmisdisordered tau in human AD brains, as well as in transgenic ratmodels of AD and related tauopathies, expressing human misdisorderedtruncated tauΔ(1-150;392-441)/3R or tauΔ(1-150;392-441)/4R. The isolatedantibodies are also capable of interfering with one or several of themultiple tau-mediated activities contributing to AD pathology,including: (i) transition from either misordered or from physiologicaltau to misdisordered tau; (ii) formation of “pathological tau” monomers,dimers, trimers, and other tau multimers;” (iii) formation of insolubletau aggregates; and (iv) promoting clearance of extracellular tau.

The disclosed invention is based, in part, on the discovery thatantibodies that specifically bind to one of four previously unidentifiedfunctional regions of tau selected from 267-KHQPGGG-273 (SEQ ID NO: 98)(within 1^(st) repeat domain of tau protein), 298-KHVPGGG-304 (SEQ IDNO: 99) (within 2^(nd) repeat domain of tau protein), 329-HHKPGGG-335(SEQ ID NO: 100) (within 3^(rd) repeat domain of tau protein), and361-THVPGGG-367 (SEQ ID NO: 101) (within 4^(th) repeat domain of tauprotein) are capable of inhibiting formation of pathological tauaggregates, and of detecting various pathological forms of tau, some ofwhich are the earliest formed in the disease (e.g., pathologicalmonomers). Hybridomas produced against human misdisordered tau II(151-391/4R), which is also referred in this application astauΔ(1-150;392-441)/4R, were screened for the production of monoclonalantibodies specific to human PHFs both by immunohistochemistry (IHC) andEnzyme-linked Immuno Assays (ELISAs). The resulting set included mousemonoclonal antibody (mAb) DC8E8, which is of the IgG1 subclass. Epitopemapping of DC8E8 revealed it to bind four previously unidentifiedepitopes on human tau. Moreover, further functional analysis of DC8E8revealed that each epitope represents a distinct functional regionwithin tau. These regions, which can now be described as novel targetsfor AD diagnosis and therapy, and thus are referred to as “therapeuticepitopes,” are comprised within 267-KHQPGGG-273 (SEQ ID NO: 98) (within1^(st) repeat domain of tau protein), 298-KHVPGGG-304 (SEQ ID NO: 99)(within 2^(nd) repeat domain of tau protein), 329-HHKPGGG-335 (SEQ IDNO: 100) (within 3^(rd) repeat domain of tau protein), and361-THVPGGG-367 (SEQ ID NO: 101) (within 4^(th) repeat domain of tauprotein). In some embodiments, one or more of the therapeutic epitopesis comprised within 268-HQPGGG-273 (SEQ ID NO: 223) (within 1^(st)repeat domain of tau protein), 299-HVPGGG-304 (SEQ ID NO: 154) (within2^(nd) repeat domain of tau protein), 330-HKPGGG-335 (SEQ ID NO: 224)(within 3^(rd) repeat domain of tau protein), and 362-HVPGGG-367 (SEQ IDNO: 154) (within 4^(th) repeat domain of tau protein). In someembodiments, at least one of the therapeutic epitopes is comprisedwithin 299-HVPGGG-304 (SEQ ID NO: 154) (within 2^(nd) repeat domain oftau protein). In some embodiments, one or more of the therapeuticepitopes is 299-HVPGGG-304 (SEQ ID NO: 154).

Indeed, DC8E8 is capable of discriminating between pathological andnormal tau proteins, suggesting that at least one of these four epitopesis conformational. In other words, DC8E8 revealed that at least one ofthe regions encompassed by each of the four therapeutic epitopes shows aconformation in pathological tau that is different from the shape(s) itassumes in intrinsically disordered tau (normal tau). DC8E8 is able tosense or detect that change in that it binds to pathological tau with ahigher affinity than it binds to physiological tau. Moreover, DC8E8binding to tau is capable of inhibiting tau-tau interactions leading upto the formation of pathological tau aggregates, as measured by DC8E8'sability to inhibit the formation of insoluble tau aggregates in vitro.For example, DC8E8 binding to normal tau is capable of preventing one ormore of the conformational/shape changes discussed above for the regionsencompassing the therapeutic epitopes.

In addition, binding of DC8E8 to normal tau at one or more of theseregions or therapeutic epitopes impedes certain other conformationalchanges elsewhere in the molecule that are needed for the production ofpathological tau. Without being bound by any specific mechanism, it iscontemplated that one or more of these epitopes/regions within tau thatare recognized by DC8E8, functions within tau as a promoter of tau-tauaggregation. For example, the structure/shape/conformation of one ormore of these epitopes influences the structure of adjacent regions,such that fixing its shape within the tau molecule by binding of DC8E8to it interferes with the adjacent region's (e.g., 274-281) ability ortendency to form beta-sheets, where beta-sheet formation is needed fortau-tau aggregation. Thus, it is contemplated that binding of DC8E8 toone of these four regions within normal tau is capable of preventing oneof the earliest pathological changes in tau identified to date: a changethat is needed to promote, or that iself promotes or allows for theformation of beta-sheets within tau. Moreover, it is also contemplatedthat binding of DC8E8 to one of these four regions withinmisdisordered/pathological tau, i.e., after one or more of the four hasalready changed to a pathological conformation, is still capable ofinhibiting pathological tau-tau aggregation at least because it stillinhibits beta-sheet formation, blocks tau-tau physical interaction, orboth.

Thus, using DC8E8 as a tool to identify novel targets or functionalregions within tau, four specific DC8E8 binding sites on tau wereassigned a role in Alzheimer's disease. This was done through therecognition that one or more of these tau sites are involved in theformation of pathological tau monomers and multimers, at least becausebinding of DC8E8 to one or more of them is capable of inhibiting thoseprocesses. Moreover, antibodies (e.g., DC8E8) that bind to one or moreof these therapeutic epitopes, are capable of promoting the clearance ofpathological tau from the extracellular environment, at least becausethey are capable of mediating the uptake and degradation of pathologicaltau by microglia, in vitro; a decrease in extracellular andintracellular tau in the brain, in vivo; or both. In other words, theseantibodies have the capacity to help reduce the damage that suchpathological forms of tau cause to the brain.

Accordingly, described herein are antibodies that specifically bind toone or more therapeutic epitopes on tau, wherein each of the therapeuticepitopes is separately located within amino acid residues267-KHQPGGG-273 (SEQ ID NO: 98) (epitope #1, within 1^(st) repeat domainof tau protein), 298-KHVPGGG-304 (SEQ ID NO: 99) (epitope #2, within2^(nd) repeat domain of tau protein), 329-HHKPGGG-335 (SEQ ID NO: 100)(epitope #3, within 3^(rd) repeat domain of tau protein), and361-THVPGGG-367 (SEQ ID NO: 101) (epitope #4, within 4^(th) repeatdomain of tau protein). In some embodiments, the therapeutic epitopes #1through 4 are comprised within 268-HQPGGG-273 (SEQ ID NO: 223) (within1^(st) repeat domain of tau protein), 299-HVPGGG-304 (SEQ ID NO: 154)(within 2^(nd) repeat domain of tau protein), 330-HKPGGG-335 (SEQ ID NO:224) (within 3^(rd) repeat domain of tau protein), and 362-HVPGGG-367(SEQ ID NO: 154) (within 4^(th) repeat domain of tau protein). Theantibodies can be monoclonal or polyclonal. Also included areantigen-binding antibody portions, antibody fragments, antibodyvariants, engineered proteins, and polymer scaffolds. These include anyprotein or peptide-containing molecule that comprises at least a portionof an immunoglobulin molecule, such as, but not limited to, at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof.

As a non-limiting example, a suitable antibody, antibody portion,fragment, or variant, as provided by the present invention, can bind toat least one of the described therapeutic epitopes. The term “antibody”also includes antibody digestion fragments, specified antibody portionsand variants thereof, including antibody mimetics, or portions ofantibodies that mimic the structure and/or function of an antibody orspecified fragment or portion thereof, including single chain antibodiesand fragments thereof. Functional fragments include antigen-bindingfragments that bind to one or more therapeutic epitopes. For example,antibody fragments capable of binding to a therapeutic epitope, include,but are not limited to Fab (e.g., by papain digestion), Fab′ (e.g., bypepsin digestion and partial reduction) and F(ab′)₂ (e.g., by pepsindigestion), facb (e.g., by plasmin digestion), pFc′ (e.g., by pepsin orplasmin digestion), Fd (e.g., by pepsin digestion, partial reduction andre-aggregation), Fv or scFv (e.g., by molecular biology techniques)fragments, are provided by the present invention. See also, William E.Paul (ed.) Fundamental Immunology, 6^(th) Edition, Lippincott Williams &Wilkins, NY, N.Y. (2008), incorporated herein in its entirety. Certainfragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as routinely known in the art, or as providedherein. Antibodies can also be produced in a variety of truncated formsusing antibody genes in which one or more stop codons have beenintroduced upstream of the natural stop site. For example, a combinationgene encoding an F(ab′)₂ heavy chain portion can be designed to includeDNA sequences encoding the CH1 domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using routine genetic engineering techniques.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25-kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxyl-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of IMGT. Alternative definitions arealso known to one of ordinary skill in the art. See, e.g. KabatSequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,or at least about 99% amino acid sequence identity to any one of SEQ IDNOs:141, 143, 152, and 153. In some embodiments, a subject antibodycomprises a light chain comprising an amino acid sequence that differsfrom any one of SEQ ID NOs:141, 143, 152, and 153 by only one, two,three, four, five, six, seven, eight, nine, or ten amino acids. Those ofordinary skill in the art can determine which amino acids in a lightchain variable region can be altered. For example, by comparing theamino acid sequences of light chain variable regions of antibodies withthe same specificity, those skilled in the art can determine which aminoacids can be altered without altering the specificity. See the EXAMPLESfor a comparison of CDR amino acid sequences of the exemplary DC8E8antibody light chain. Furthermore, whether the specificity is alteredcan be determined using an antigen binding assay. In some embodiments, asubject antibody comprises a light chain comprising an amino acidsequence as set forth in any one of SEQ ID NOs:141, 143, 152, and 153.

In some embodiments, a subject antibody comprises a heavy chaincomprising an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,or at least about 99% amino acid sequence identity to any one of SEQ IDNOs:138, 140, 147, and 148. In some embodiments, a subject antibodycomprises a heavy chain comprising an amino acid sequence that differsfrom any one of SEQ ID NOs:138, 140, 147, and 148 by only one, two,three, four, five, six, seven, eight, nine, or ten amino acids. Those ofordinary skill in the art can determine which amino acids in a heavychain variable region can be altered. For example, by comparing theamino acid sequences of heavy chain variable regions of antibodies withthe same specificity, those skilled in the art can determine which aminoacids can be altered without altering the specificity. See, e.g., FIGS.3E and 25B for a comparison of CDR amino acid sequences of exemplaryDC8E8 antibody heavy chain. Furthermore, whether the specificity isaltered can be determined using an antigen binding assay. In someembodiments, a subject antibody comprises a heavy chain comprising anamino acid sequence as set forth in any one of SEQ ID NOs:138, 140, 147,and 148.

In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:141 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:138. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:141 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:140. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:141 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:147. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:141 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:148. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:143 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:138. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:143 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:140. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:143 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:147. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:143 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:148. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:152 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:138. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:152 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:140. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:152 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:147. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:152 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:148. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:153 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:138. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:153 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:140. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:153 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:147. In some embodiments, a subject antibody comprises a light chaincomprising an amino acid sequence as set forth in SEQ ID NO:153 and aheavy chain comprising an amino acid sequence as set forth in SEQ IDNO:148.

In some embodiments, the subject antibody comprises a light chainvariable region comprising at least one, at least two, or three CDRschosen from SEQ ID NOs. 117-119. In some embodiments, the subjectantibody comprises a heavy chain variable region comprising at leastone, at least two, or three CDRs chosen from SEQ ID NOs. 120-122. Alsoprovided are embodiments in which any one of these six CDRs is alteredas described in EXAMPLE 14. In some embodiments, at least one of thealtered CDRs in the light chain is chosen from SEQ ID NO: 247 for CDR1,SEQ ID NO: 253 for CDR2, and any one of SEQ ID NOs: 255, 257, 258, 259,and 260 for CDR3. In some embodiments, at least one of the altered CDRsin the heavy chain is chosen from SEQ ID NO: 261, or SEQ ID NO: 262 forCDR1, SEQ ID NO: 264, or SEQ ID NO: 265 for CDR2, and SEQ ID NO: 266,SEQ ID NO: 267, or SEQ ID NO: 269 for CDR3.

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547-1553 (1992). Production of bispecificantibodies can be a relatively labor intensive process compared withproduction of conventional antibodies and yields and degree of purityare generally lower for bispecific antibodies. Bispecific antibodies donot exist in the form of fragments having a single binding site (e.g.,Fab, Fab′, and Fv).

The invention does not relate to antibodies in natural form, i.e., theyare not taken from their natural environment but are isolated andobtained by purification from natural sources, or obtained by geneticrecombination or chemical synthesis, and thus they can carry unnaturalamino acids. Thus, as used herein, the twenty conventional amino acidsand their abbreviations follow conventional usage. See Immunology-ASynthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., SinauerAssociates, Sunderland, Mass. (1991)), which is incorporated herein byreference. Stereoisomers (e.g., D-amino acids, Nle, Nva, Cha, Orn, Hle,Chg, Hch, or Har) of the twenty conventional amino acids, unnaturalamino acids such as .alpha.-, .alpha.-disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids can alsobe suitable components for polypeptides of the present invention.Examples of unconventional amino acids include (i.e., are not limitedto): 4-hydroxyproline, gamma.-carboxyglutamate,epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, .sigma.-N-methylarginine, and other similar amino acidsand imino acids (e.g., 4-hydroxyproline). In the polypeptide notationused herein, the left-hand direction is the amino terminal direction andthe right-hand direction is the carboxy-terminal direction, inaccordance with standard usage and convention

Similarly, the present disclosure does not relate to nucleotidesequences in their natural chromosomal environment, i.e., in a naturalstate. The sequences of the present invention have been isolated andpurified, i.e., they were sampled directly or indirectly, for example bya copy, their environment having been at least partially modified.Isolated nucleic acids obtained by recombinant genetics, by means, forexample, of host cells, or obtained by chemical synthesis are alsoprovided

In relation to this disclosure, the “percentage identity” between twosequences of nucleic acids or amino acids means the percentage ofidentical nucleotides or amino acid residues between the two sequencesto be compared, obtained after optimal alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly along their length. The comparison of two nucleicacid or amino acid sequences is traditionally carried out by comparingthe sequences after having optimally aligned them, said comparison beingable to be conducted by segment or by using an “alignment window”.Optimal alignment of the sequences for comparison can be carried out, inaddition to comparison by hand, by means of the local homology algorithmof Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of thelocal homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol.48:443], by means of the similarity search method of Pearson and Lipman(1988) [Proc. Natl. Acad. Sci. USA 85:2444] or by means of computersoftware using these algorithms (GAP, BESTFIT, FASTA and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis., or by the comparison software BLAST NR orBLAST P).

The percentage identity between two nucleic acid or amino acid sequencesis determined by comparing the two optimally-aligned sequences in whichthe nucleic acid or amino acid sequence to compare can have additions ordeletions compared to the reference sequence for optimal alignmentbetween the two sequences. Percentage identity is calculated bydetermining the number of positions at which the amino acid nucleotideor residue is identical between the two sequences, for example betweenthe two complete sequences, dividing the number of identical positionsby the total number of positions in the alignment window and multiplyingthe result by 100 to obtain the percentage identity between the twosequences.

For example, the BLAST program, “BLAST 2 sequences” (Tatusova et al.,“Blast 2 sequences—a new tool for comparing protein and nucleotidesequences”, FEMS Microbiol., 1999, Lett. 174:247-250) available on thesite http://www.ncbi.nlm.nih.gov/gorf/b12.html, can be used with thedefault parameters (notably for the parameters “open gap penalty”: 5,and “extension gap penalty”: 2; the selected matrix being for examplethe “BLOSUM 62” matrix proposed by the program); the percentage identitybetween the two sequences to compare is calculated directly by theprogram.

For the amino acid sequence exhibiting at least 80%, for example 85%,90%, 95% and 98% identity with a reference amino acid sequence,preferred examples include those containing the reference sequence,certain modifications, notably a deletion, addition or substitution ofat least one amino acid, truncation or extension. In the case ofsubstitution of one or more consecutive or non-consecutive amino acids,substitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. Here, the expression “equivalentamino acids” is meant to indicate any amino acids likely to besubstituted for one of the structural amino acids without howevermodifying the biological activities of the corresponding antibodies andof those specific examples defined below.

Equivalent amino acids can be determined either on their structuralhomology with the amino acids for which they are substituted or on theresults of comparative tests of biological activity between the variousantibodies likely to be generated. As a non-limiting example, the tablebelow summarizes the possible substitutions likely to be carried outwithout resulting in a significant modification of the biologicalactivity of the corresponding modified antibody; inverse substitutionsare naturally possible under the same conditions.

Original residue Substitution(s) Ala (A) Val, Gly, Pro Arg (R) Lys, HisAsn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) AlaHis (H) Arg Ile (I) Leu Leu (L) Ile, Val, Met Lys (K) Arg Met (M) LeuPhe (F) Tyr Pro (P) Ala Ser (S) Thr, Cys Thr (T) Ser Trp (W) Tyr Tyr (Y)Phe, Trp Val (V) Leu, Ala

The invention provides an antibody produced by the mouse hybridoma cellline deposited with the American Type Culture Collection, (ATCC, 10801University Blvd, Manassas, Va., USA) on Jul. 13, 2011, with the ATCCPatent Deposit Designation PTA-11994 (issued Jul. 29, 2011), asdescribed in Examples 1-2. Other suitable antibodies can be produced bya cell line, a mixed cell line, an immortalized cell or clonalpopulation of immortalized cells, as known in the art. See, e.g.,Ausubel et al. (Ed.), Current Protocols in Molecular Biology, (JohnWiley & Sons, Inc., New York, N.Y. (1987-2001)); Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition, (Cold SpringHarbor, N.Y. (1989)) and Sambrook et al., Molecular Cloning—A LaboratoryManual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 2000 (collectively, “Sambrook”); Harlow and Lane,Antibodies, A Laboratory Manual, (Cold Spring Harbor, N.Y. (1989));Colligan, et al. (Eds.), Current Protocols in Immunology, (John Wiley &Sons, Inc., N.Y. (1994-2001)); Colligan et al., Current Protocols inProtein Science, (John Wiley & Sons, NY, N.Y., (1997-2001)), eachentirely incorporated herein by reference.

In one approach for producing the antibodies provided by the invention,a hybridoma is produced by fusing a suitable immortal cell line (e.g., amyeloma cell line) with one of a variety of antibody-producing cells.Suitable immortal cell lines include, but not limited to, Sp2/0,Sp2/0-AG14, P3/NS1/Ag4-1, NSO, P3X63Ag8.653, MCP-11, S-194,heteromyelomas, fusion products thereof, or any cell or fusion cellderived therefrom, or any other suitable cell line as known in the art,and/or commercially available for this purpose (e.g., ATCC). Suitableantibody producing cells include, but are not limited to, isolated orcloned spleen, peripheral blood, lymph, tonsil, or other immune or Bcell containing cells, or any other cells expressing heavy or lightchain constant or variable or framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination of the same. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, Chapter 2,entirely incorporated herein by reference.

Other approaches for producing the antibodies of the various embodimentsdescribed above include, but are not limited to, methods that selectrecombinant antibodies from peptide or protein libraries, includingthose commercially available from Cambridge antibody Technologies,Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, Del.; Biovation,Aberdeen, Scotland, UK; Biolnvent, Lund, Sweden; Dyax Corp., Enzon,Affymax/Biosite; Xoma, Berkeley, Calif.; Ixsys; Applied MolecularEvolution; and the like; methods that rely upon immunization oftransgenic animals that are capable of producing selectable sets ofhuman antibodies (generally, these mice comprise at least one transgenecomprising DNA from at least one human immunoglobulin locus that isfunctionally rearranged, or which can undergo functional rearrangement;the endogenous immunoglobulin loci in such mice can be disrupted ordeleted to eliminate the capacity of the animal to produce antibodiesencoded by endogenous genes); selection methods including ribosomedisplay, single cell antibody producing technologies (e.g., selectedlymphocyte antibody method (“SLAM”)), and B-cell selection; subtractiveimmunization using cyclophosamide treatment; as well as any othermethods routine in the art, including, but not limited to, thosedescribed in US Published Application No. 2005/0142609, which methodsare entirely incorporated herein by reference).

In some embodiments, the antibodies are optimized full-lengthantibodies, chimeric or humanized, which can be produced by any one or acombination of known techniques, as listed and exemplified in, forexample, Chapters 3, 4, and 5, of “Business Insights, PreclinicalDevelopment of Monoclonal Antibodies and Related Biologicals-Emergingtechnologies and new therapeutic candidates, by James Shirvill, 2010,”the entire contents of which is incorporated by reference, such as: CDRgrafting, such as UBC's SLAM technology, PDL's SMART technology, AranaTherapeutics plc's Superhumanization, Framework patching, techniques formaking composit human antibodies, BioAtla LLC's ATLAb platform,humaneering, Mutational Lineage Guided (MLG) strategies, deimmunisationstrategies, humanation strategies, human engineering (e.g., XOMA's HEtechnology), FcX, Biolex Therapeutics Inc (Pittsboro, N.C., US) LEXsystem, Potelligent approaches (e.g., BioWa), Complegent technology,BestMAb, ImmunoBody, EB66, Synageva Expression Platforms, Xencor Inc.XmAb, Sugar Engineered Antibodies (e.g., Seattle Genetics Inc (Bothell,Wash., US)), “Wox” (tryptophan oxidized) antibodies (e.g., InNexusBiotechnology Inc (Vancouver, BC, Canada)); and the like. In someembodiments, the antibodies are fully human monoclonal antibodies, andcan be produced by one or a combination of technology platforms, aslisted and exemplified in, for example, Chapter 4 of “Business Insights,Preclinical Development of Monoclonal Antibodies and RelatedBiologicals-Emerging technologies and new therapeutic candidates, byJames Shirvill, 2010,” and including, but not limited to: phage display(e.g., PDL, Dyax Corp; Cambridge, Mass., US); Molecule Based AntibodyScreening (MBAS) (e.g., Affitech A/S, described in, e.g., EP0547201 andU.S. Pat. No. 6,730,483); cell based antibody selection (CBAS)platforms; Human Combinatorial Antibody Libraries (HuCAL; e.g.,MorphoSys AG); MAbstract platforms (e.g., Crucell NV), including thosewith the PER.C6 cell line; Adimab platforms; XenoMouse; UltiMAbplatforms; SEBVI platforms; Veloclmmune platforms, Open MonoclonalTechnology platforms, Xenerex platforms; Cloning the Human Responseplatforms (e.g., IQ Therapeutics) and “Instant Immunity antibodies;”Viventia platforms (e.g., Fusogenics, UnLock, ImmunoMine); “NaturalHuman Antibodies” platforms (e.g., OncoMab, Patrys, Acceptys); MablgX(e.g., Kenta Biotech); Reverse Translational Medicine platforms (e.g.,Neuimmune Therapeutics); I-STAR (e.g., Theraclone Sciences); CellSpot(e.g., Trellis Bioscience); iBioLaunch (e.g., iBio Inc.); and the like.

In some embodiments, the antibodies are modified by linking them tonon-antibody agents, using one or more of the technology platforms andmethods as described in Chapter 5 of “Business Insights, PreclinicalDevelopment of Monoclonal Antibodies and Related Biologicals-Emergingtechnologies and new therapeutic candidates, by James Shirvill, 2010,”including: antibody drug conjugate (e.g., ADC, Seattle Genetics);targeted antibody payload (TAP; Immunogen Inc); Probodies (e.g., CytomXTherapeutics); antibody cloaking (e.g., BioTransformations); targetedphotodynamic therapy (e.g., PhotoBiotics; AlbudAb (e.g., GSK); hyFc(e.g., Genexine); Ligand traps (e.g., BioLogix); CovX-Body (e.g., CovX);Dynamic Cross-Linking (e.g., InNexus Biotechnology); LEC Technology(e.g., Pivotal BioSciences, Morphotek); and the like.

In some embodiments, the antibody or its encoding cDNAs can be furthermodified. Thus, in a further embodiment, the invention provides methodsof producing the antibodies of the various embodiments, wherein themethods comprise any one of the step(s) of producing a chimericantibody, humanized antibody, or an analog of any one of those. In someembodiments, the production of chimeric antibodies is as described ininternational application WO89/09622. Methods for the production ofhumanized antibodies are described in, e.g., U.S. Pat. No. 6,548,640 orCanadian Patent No. 1340879 (CDR grafting).

In addition, the antibody or its encoding cDNAs can be further modified.Thus, in a further embodiment, the invention provides methods comprisingany one of the step(s) of producing a single-chain antibody,Fab-fragment, bi-specific antibody, fusion antibody, labeled antibody oran analog of any one of those. As discussed above, the antibody of theinvention can exist in a variety of forms besides complete antibodies;including, for example, Fv, Fab and F(ab)2, as well as in single chains.See e.g. international application WO88/09344. Furthermore, diabodiesand V-like domain binding molecules are well-known to the person skilledin the art; see, e.g. U.S. Pat. No. 7,166,697.

In some embodiments, the antibodies (e.g., DC8E8) are modified or serveas the basis for making binding molecules with one or more of theantigen-binding properties described for the DC8E8 antibody. Thesebinding proteins can be made by one or more of the techniques listed andexemplified in, for example, Chapter 6 of “Business Insights,Preclinical Development of Monoclonal Antibodies and RelatedBiologicals-Emerging technologies and new therapeutic candidates, byJames Shirvill, 2010,” including:Fab, TetraMABs (e.g., GalileoOncologics); scFv; Immuna (e.g., ESBA Tech AG); [scFv]2, includingbinding molecules that bind any two of the four therapeutic epitopes ofDC8E8; BiTE (Affitech, Micromet AG); Avibodies (e.g., Avipep Pty);TandAb (e.g., Affimed Therapeutics); Flexibody (e.g., Affimed); V-NAR(e.g., AdAlta); Nanobody (Ablynx NV); Domain Antibodies (e.g., DiversysLtd. GSK, U.S. Pat. No. 6,248,516 and EP0368684); Heteropolymer (e.g.,Elusys Therapeutics Inc.); Unibody (e.g., GenMab NS); Domain ExchangedAntibodies (e.g., Calmune Corporation, Science. 2003 June 27;300(5628):2065-71); Small Modular ImmunoPharmaceuticals (SMIP) andSCORPION molecules (e.g., Trubion Pharmaceuticals); Dual Variable DomainImmunoglobulin, DVD-Ig (Abbott Laboratories); and the like.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. See, e.g., the EXAMPLES provided furtherbelow. Methods for introducing such modifications in the DNA sequenceunderlying the amino acid sequence of an immunoglobulin chain are knownto the person skilled in the art. See, e.g., Sambrook (supra) andAusubel (Supra). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment or removal ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as an immunostimulatoryligand at the carboxyl terminus. See, e.g., international applicationWO00/30680 for corresponding technical details, incorporated herein byreference in its entirety.

In one embodiment, the invention relates to a method for the productionof an antibody or a binding fragment or immunoglobulin chain(s) thereof,the method comprising

-   -   (a) culturing a cell as described above; and    -   (b) isolating said antibody or binding fragment or        immunoglobulin chain(s) thereof from the culture.

In some embodiments, the isolation comprises contacting theantibody-containing sample with one of the peptides provided by theinvention, to which the antibody binds to.

The transformed hosts can be grown in fermentors and cultured accordingto techniques known in the art to achieve optimal cell growth. Onceexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like; see, Scopes, “Protein Purification”,Springer Verlag, N.Y. (1982). The antibody or its correspondingimmunoglobulin chain(s) can then be isolated from the growth medium,cellular lysates, or cellular membrane fractions. The isolation andpurification of the, e.g., recombinantly expressed antibodies orimmunoglobulin chains provided by the invention can be done by anyconventional means such as, for example preparative chromatographicseparations and immunological separations, like those involving the useof monoclonal or polyclonal antibodies directed against the constantregion of the antibody of the invention.

Substantially pure immunoglobulins of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity mostpreferred, for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the antibodies can then be used therapeutically(including extracorporally) or in developing and performing assayprocedures.

The invention also provides antibodies coupled to other moieties forpurposes such as drug targeting and imaging applications. Such couplingcan be conducted chemically after expression of the antibody to site ofattachment or the coupling product can be engineered into the antibodyof the invention at the DNA level. The DNAs are then expressed in asuitable host system, and the expressed proteins are collected andrenatured, if necessary.

The present invention also involves a method for producing cells capableof expressing an antibody of the invention or its correspondingimmunoglobulin chain(s) comprising genetically engineering cells withthe polynucleotide or with the vector of the invention. The cellsobtainable by the method of the invention can be used, for example, totest the interaction of the antibody of the invention with its antigen.

The invention provides also antibody-producing cell lines andrecombinant cells, as a source of the antibodies provided by the presentinvention. The present invention further relates to diagnostic assaysand kits that comprise the antibodies provided by the invention or anequivalent binding molecule and to therapeutic methods based thereon.

The invention also provides methods for producing antibodies that arecapable of competing with DC8E8 and are also capable of inhibitingpathological tau-tau interactions. Those antibodies can be screened bytheir ability to sufficiently compete with DC8E8 for binding to tau andbinding to one, two, three, or all four of the “therapeutic epitopes”identified herein.

The present invention also relates to polynucleotides encoding one ormore of the antibody-based agents provided by the invention. In certaincases, the nucleotide for example encodes at least the binding domain orvariable region of an immunoglobulin chain of the antibodies describedabove. Typically, said variable region encoded by the polynucleotidecomprises at least one complementarity determining region (CDR) of theVH and/or VL of the variable region of the said antibody. The personskilled in the art knows that each variable domain (the heavy chain VHand light chain VL) of an antibody comprises three hypervariableregions, sometimes called complementarity determining regions or “CDRs”flanked by four relatively conserved framework regions or “FRs” andrefer to the amino acid residues of an antibody which are responsiblefor antigen-binding. According to the Kabat numbering system, thehypervariable regions or CDRs of the human IgG subtype of antibodycomprise amino acid residues from residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain as described by Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)and/or those residues from a hypervariable loop, i.e. residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain as described by Chothia et al., J. Mol. Biol. 196 (1987),901-917. In the IMGT unique numbering system, the conserved amino acidsalways have the same position, for instance cysteine 23 (1st-CYS),tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cystein 104(2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). See, e.g.,Lefranc M.-P., Immunology Today 18, 509 (1997); Lefranc M.-P., TheImmunologist, 7, 132-136 (1999); Lefranc, M.-P., Pommie, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. andLefranc, Dev. Comp. Immunol., 27, 55-77 (2003). The IMGT uniquenumbering provides a standardized delimitation of the framework regions(FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104and FR4-IMGT: 118 to 128) and of the complementarity determiningregions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to117. The IMGT unique numbering is used in 2D graphical representations,designated as IMGT Colliers de Perles. See, e.g., Ruiz, M. and Lefranc,M.-P., Immunogenetics, 53, 857-883 (2002); Kaas, Q. and Lefranc, M.-P.,Current Bioinformatics, 2, 21-30 (2007). It is also used forrepresenting 3D structures. See, e.g., IMGT/3Dstructure-DB Kaas, Q.,Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data.Nucl. Acids. Res., 32, D208-D210 (2004). Framework or FR residues arethose variable domain residues other than and bracketing thehypervariable regions.

Accordingly, the invention also relates to an isolated nucleic acidcharacterized in that it is selected among the following nucleic acids(including any degenerate genetic code):

-   -   a) a nucleic acid, DNA or RNA, coding for an antibody according        to the invention;    -   b) a nucleic acid complementary to a nucleic acid as defined in        a);    -   c) a nucleic acid of at least 18 nucleotides capable of        hybridizing under highly stringent conditions with at least one        of the CDRs chosen from SEQ ID NOs. 117-122 and SEQ ID NOs. 247,        253, 255, 257-259, 122, 261, 262, 264, 265-267, and 269; and    -   d) a nucleic acid of at least 18 nucleotides capable of        hybridizing under highly stringent conditions with at least the        light chain of nucleic acid sequence SEQ ID 165 and/or the heavy        chain of nucleic acid sequence SEQ ID No. 170, or a sequence        with at least 80%, for example 85%, 90%, 95% and 98% identity        after optimal alignment with sequences SEQ ID Nos. 165 and/or        SEQ ID 170, for example with at least one of the CDRs therefrom        according to the IMGT numbering.

Nucleic sequences exhibiting a percentage identity of at least 80%, forexample 85%, 90%, 95% and 98%, after optimal alignment with a preferredsequence, means nucleic sequences exhibiting, with respect to thereference nucleic sequence, certain modifications such as, inparticular, a deletion, a truncation, an extension, a chimeric fusionand/or a substitution, notably punctual. In some embodiments, these aresequences which code for the same amino acid sequences as the referencesequence, this being related to the degeneration of the genetic code, orcomplementarity sequences that are likely to hybridize specifically withthe reference sequences, for example under highly stringent conditions,notably those defined below.

Hybridization under highly stringent conditions means that conditionsrelated to temperature and ionic strength are selected in such a waythat they allow hybridization to be maintained between twocomplementarity DNA fragments. On a purely illustrative basis, thehighly stringent conditions of the hybridization step for the purpose ofdefining the polynucleotide fragments described above are advantageouslyas follows.

DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)prehybridization at 42° C. for three hours in phosphate buffer (20 mM,pH 7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15 MNaCl+0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate(SDS), 10×Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2)primary hybridization for 20 hours at a temperature depending on thelength of the probe (i.e.: 42° C. for a probe >100 nucleotides inlength) followed by two 20-minute washings at 20° C. in 2×SSC+2% SDS,one 20-minute washing at 20° C. in 0.1×SSC+0.1% SDS. The last washing iscarried out in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for aprobe >100 nucleotides in length. The highly stringent hybridizationconditions described above for a polynucleotide of defined size can beadapted by a person skilled in the art for longer or shorteroligonucleotides, according to the procedures described in Sambrook, etal. (Molecular cloning: a laboratory manual, Cold Spring HarborLaboratory; 3rd edition, 2001).

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method; see, for example, Pope M E,Soste M V, Eyford B A, Anderson N L, Pearson T W. (2009) J ImmunolMethods. 341(1-2):86-96, and methods described herein. The measuredaffinity of a particular antibody-antigen interaction can vary ifmeasured under different conditions, e.g., salt concentration, pH. Thus,measurements of affinity and other antigen-binding parameters, e.g., Ksub D, IC50, are for example made with standardized solutions ofantibody and antigen, and a standardized buffer.

The invention also provides that the variable domain of the antibodyhaving the above-described variable domain can be used for theconstruction of other polypeptides or antibodies of desired specificityand biological function. Thus, the present invention also encompassespolypeptides and antibodies comprising at least one CDR of theabove-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art willappreciate that using the variable domains or CDRs described hereinantibodies can be constructed according to methods known in the art,e.g., as described in European patent applications EP 0 451 216 A1 andEP 0 549 581 A1. Furthermore, the person skilled in the art knows thatbinding affinity can be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs,as defined by Kabat. Thus, the present invention also relates toantibodies wherein one or more of the mentioned CDRs comprise one ormore, for example not more than two amino acid substitutions. In someembodiments, the antibody of the invention comprises in one or both ofits immunoglobulin chains two or all three CDRs of the variable regionsas set forth in FIGS. 3B and 3E. In some embodiments, the antibody ofthe invention comprises in one or both of its immunoglobulin chains twoor all three CDRs as set forth in FIG. 25B.

The polynucleotides or nucleic acids encoding the above describedantibodies can be, e.g., DNA, cDNA, RNA or synthetically produced DNA orRNA or a recombinantly produced chimeric nucleic acid moleculecomprising any of those polynucleotides either alone or in combination.In some embodiments, the polynucleotide is part of a vector. Suchvectors can comprise further genes such as marker genes which allow forthe selection of said vector in a suitable host cell and under suitableconditions.

In some embodiments, the polynucleotide is operatively linked to one ormore expression control sequences, allowing expression in prokaryotic oreukaryotic cells. Expression of said polynucleotide comprisestranscription of the polynucleotide into a translatable mRNA. Regulatoryelements ensuring expression in eukaryotic cells, for example mammaliancells, are known to those skilled in the art. They usually compriseregulatory sequences ensuring initiation of transcription and optionallypoly-A signals ensuring termination of transcription and stabilizationof the transcript. Additional regulatory elements can includetranscriptional as well as translational enhancers, and/or naturallyassociated or heterologous promoter regions.

In this respect, the person skilled in the art will appreciate that thepolynucleotides encoding at least the variable domain of the lightand/or heavy chain can encode the variable domains of bothimmunoglobulin chains or only one. Likewise, said polynucleotides can beunder the control of the same promoter or can be separately controlledfor expression. Possible regulatory elements permitting expression inprokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoterin E. coli, and examples for regulatory elements permitting expressionin eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or theCMV-, SV40-, RSV-promoter, CMV-enhancer, SV40-enhancer or a globinintron in mammalian and other animal cells.

Beside elements that are responsible for the initiation oftranscription, such regulatory elements can also comprise transcriptiontermination signals, such as the SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. Furthermore, depending on theexpression system used, leader sequences capable of directing thepolypeptide to a cellular compartment or secreting it into the mediumcan be added to the coding sequence of the polynucleotides and are knownin the art. The leader sequence(s) is (are) assembled in appropriatephase with translation, initiation and termination sequences, andoptionally, a leader sequence capable of directing secretion oftranslated protein, or a portion thereof, into the periplasmic space orextracellular medium. In some embodiments, the heterologous sequence canencode a fusion protein including a C- or N-terminal identificationpeptide imparting desired characteristics, e.g., stabilization orsimplified purification of expressed recombinant product. In thiscontext, suitable expression vectors are known in the art, and include,without limitation, the Okayama-Berg cDNA expression vector pcDV1(Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), and pSPORT1(GIBCO BRL).

In some embodiments, the expression control sequences can be eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells, but control sequences for prokaryotic hosts canalso be used. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the immunoglobulin light chains, heavy chains,light/heavy chain dimers or intact antibodies, binding fragments orother immunoglobulin forms can follow. See, e.g., Beychok, Cells ofImmunoglobulin Synthesis, Academic Press, N.Y., (1979).

Furthermore, the invention provides vectors, particularly plasmids,cosmids, viruses and bacteriophages used conventionally in geneticengineering that comprise a polynucleotide encoding a variable domain ofan immunoglobulin chain of an antibody of the invention; optionally incombination with a polynucleotide of the invention that encodes thevariable domain of the other immunoglobulin chain of an antibody of theinvention. In some embodiments, said vector is an expression vectorand/or a gene transfer or targeting vector. Expression vectors derivedfrom viruses such as retroviruses, vaccinia virus, adeno-associatedvirus, herpes viruses, or bovine papilloma virus, can be used fordelivery of the polynucleotides or vector of the invention into targetedcell population. Any methods that are known to those skilled in the artcan be used to construct recombinant viral vectors. See, for example,the techniques described in Sambrook (supra) and Ausubel (supra).Alternatively, the polynucleotides and vectors provided by the inventioncan be reconstituted into liposomes for delivery to target cells. Thevectors containing the polynucleotides provided by the invention (e.g.,the heavy and/or light variable domain(s) of the immunoglobulin chainsencoding sequences and expression control sequences) can be transferredinto the host cell by known methods, which vary depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment orelectroporation can be used for other cellular hosts.

The present invention furthermore relates to host cells transformed witha polynucleotide or vector provided by the invention. The host cell canbe a prokaryotic or eukaryotic cell. The polynucleotide or vector thatis present in the host cell can either be integrated into the genome ofthe host cell or it can be maintained extrachromosomally. The host cellcan be any prokaryotic or eukaryotic cell, such as a bacterial, insect,fungal, plant, animal or human cell. Preferred fungal cells are, forexample, those of the genus Saccharomyces, in particular those of thespecies S. cerevisiae. Depending upon the host employed in a recombinantproduction procedure, the antibodies or immunoglobulin chains encoded bythe polynucleotide of the present invention can be glycosylated or canbe non-glycosylated. Certain antibodies provided by the invention, orthe corresponding immunoglobulin chains, can also include an initialmethionine amino acid residue. A polynucleotide of the invention can beused to transform or transfect the host using any of the techniquescommonly known to those of ordinary skill in the art. Furthermore,methods for preparing fused, operably linked genes and expressing themin, e.g., mammalian cells and bacteria are well-known in the art. See,e.g., Sambrook. The genetic constructs and methods described therein canbe utilized for expression of the antibodies provided by the invention,or their corresponding immunoglobulin chains, in eukaryotic orprokaryotic hosts. In general, expression vectors containing promotersequences which facilitate the efficient transcription of the insertedpolynucleotide are used in connection with the host. The expressionvector typically contains an origin of replication, a promoter, and aterminator, as well as specific genes which are capable of providingphenotypic selection of the transformed cells. Suitable source cells forthe DNA sequences and host cells for immunoglobulin expression andsecretion can be obtained from a number of sources, such as the AmericanType Culture Collection (“Catalogue of Cell Lines and Hybridomas,” Fifthedition (1985) Manassas, Va., U.S.A., and other available version,incorporated herein by reference). Furthermore, transgenic animals, forexample mammals, comprising cells of the invention can be used for thelarge scale production of the antibody of the invention.

Additionally, the present invention encompasses small peptides includingthose containing a binding molecule as described above, for examplecontaining the CDR3 region of the variable region of any one of thementioned antibodies, in particular CDR3 of the heavy chain since it hasfrequently been observed that, for certain antibodies, the heavy chainCDR3 (HCDR3) is the region having a greater degree of variability and apredominant participation in antigen-antibody interaction. Such peptidescan be synthesized or produced by recombinant means to produce a bindingagent useful according to the invention. Such methods are known to thoseof ordinary skill in the art. Peptides can be synthesized for example,using automated peptide synthesizers which are commercially available.The peptides can also be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

The above described fusion proteins can further comprise a cleavablelinker or cleavage site for proteinases, which can be called spacermoieties. These spacer moieties, in turn, can be either insoluble orsoluble (Diener et al., Science 231 (1986), 148) and can be selected toenable drug release from the antibody at the target site. Examples oftherapeutic agents which can be coupled to the antibodies of the presentinvention for immunotherapy are drugs, radioisotopes, lectins, andtoxins. The drugs that can be conjugated to the antibodies and antigensof the present invention include compounds which are classicallyreferred to as drugs such as mitomycin C, daunorubicin, and vinblastine.In using radioisotopically conjugated antibodies or antigens of theinvention for, e.g., immunotherapy, certain isotopes can be morepreferable than others depending on such factors as leukocytedistribution as well as isotype stability and emission. Depending on theautoimmune response, some emitters can be preferable to others. Ingeneral, alpha and beta particle emitting radioisotopes are preferred inimmunotherapy. In certain preferred cases, the radioisotopes are shortrange, high energy alpha emitters such as ²¹²Bi. Examples ofradioisotopes which can be bound to the antibodies or antigens of theinvention for therapeutic purposes are ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi,²¹²At, ²¹¹Pb, ⁴⁷Sc, ¹⁰⁹Pd and ¹⁸⁸Re. In certain cases, the radiolabel is⁶⁴Cu. Other therapeutic agents which can be coupled to the antibody orantigen of the invention, as well as ex vivo and in vivo therapeuticprotocols, are known, or can be ascertained, by those of ordinary skillin the art. Wherever appropriate, the person skilled in the art can usea polynucleotide of the invention encoding (and as the source for) anyone of the above described antibodies, antigens, or the correspondingvectors, instead of the proteinaceous material itself.

The invention also relates to the use of a binding molecule or anantibody, as provided herein, for the preparation of a composition foruse in vivo for suppressing formation of, or for otherwise reducing thelevels of, misdisordered and/or misordered tau in a subject; or forextra-corporeal extraction of pathological tau compounds or theirprecursors from body fluids. These methods can be used for improvingcognition or slowing or reversing cognitive decline associated withdiseases. The antibody or binding molecules provided by the invention,or chemical derivatives thereof, can be administered directly to theblood or CSF and sequestered in a subsequent step by affinity capturefrom the blood or CSF, whereby misordered and misdisordered tau issequestered together with the aforementioned binding molecule. Hence,the present invention also relates to a method of treating or preventingthe onset or progression of Alzheimer's disease or related tauopathiesin a subject comprising removing blood or CSF from the body of thesubject, subjecting the blood and CSF and returning to the subject theblood and CSF, respectively, so obtained.

Molecules and particles with an antibody, peptide, or bindingmolecule/protein of the invention also have diagnostic utility. Theinvention provides antibodies that recognize and distinguish distinctforms of tau protein that are present at distinct stages of Alzheimer'sdisease. These antibodies are capable of detecting tau (and its variousconformational changes) both in vitro and in vivo. The antibodies candistinguish between physiological and pathological tau in a variety ofassays, including biochemical, immunoprecipitation, ELISA, Westernblotting, and immunohistochemistry assays (e.g., fresh, fixed, frozen,paraffin-embeded), as well as in vivo imaging using, e.g., radiolabeledDC8E8 (including fragments of DC8E8 such as single chain DC8E8), whichdistinguishes physiological from pathological tau (see EXAMPLES). Theyare capable of doing so in both solid and fluid (e.g., blood, plasma,CSF, homogenates) animal (e.g., rodents, humans) samples and biopsies.Some of these detection assays are described in the EXAMPLES below.Other routine methods for detecting proteins are known to those of skillin the art, and thus can be routinely adapted to the antibodies,peptides, and tau-binding molecules, provided by the invention. Theantibodies of the present invention can be labeled (e.g., fluorescent,radioactive, enzyme, nuclear magnetic, heavy metal) and used to detectspecific targets in vivo or in vitro including immunochemistry-likeassays in vitro (see, e.g., the EXAMPLES described below). Also, invivo, they could be used in a manner similar to nuclear medicine imagingtechniques to detect tissues, cells, or other material havingmisdisordered tau and deposits thereof. Targeting intracellular andextracellular misdisordered tau and neurofibrillary lesions withdiagnostic imaging probes detectable by MRI or PET would provide abiological marker for a more definitive premortem diagnosis of AD, aswell as means for monitoring the efficacy of therapies targeting tauprotein. Thus, the invention provides for the use of the antibodiesdescribed herein for the preparation of a composition for, and inmethods of, tau detection and/or targeting a diagnostic agent topathological tau and neurofibrillary lesions of the brain for ADdiagnosis. These compositions and methods can be used as part of atreatment protocol for AD and related tauopathies.

The invention provides antibodies suitable for use in immunoassays inwhich they can be utilized in liquid phase or bound to a solid phasecarrier. Examples of immunoassays which can utilize the antibodies ofthe invention are competitive and non-competitive immunoassays in eithera direct or indirect format. Examples of such immunoassays are theradioimmunoassay (RIA), the sandwich (immunometric assay), flowcytometry and the Western blot assay. The antibodies of the inventioncan be bound to one of many different carriers and used to isolate cellsspecifically bound thereto. Examples of known carriers include glass,polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The carrier can beeither soluble or insoluble for the purposes of the invention. There aremany different labels and methods of labeling known to those of ordinaryskill in the art.

Examples of the types of labels that can be used in the presentinvention include enzymes, radioisotopes and radionuclides, colloidalmetals, fluorescent compounds, chemiluminescent compounds, biotinylgroups, predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags), andchemi/electrochemi/bioluminescent compounds. The enzymes includeperoxidase (e.g., HRP), luciferase, alkaline phosphatase,α-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase,acetylcholinesterase, lysozyme, malate dehydrogenase or glucose-6phosphate dehydrogenase. Alternatively, the label is biotin,digoxigenin, or 5-bromo-desoxyuridine. Fluorescent labels can be alsocombined with the antibodies and tau-binding proteins provided by theinvention, including rhodamine, lanthanide phosphors, fluorescein andits derivatives, fluorochromes, rhodamine and its derivatives, greenfluorescent protein (GFP), Red Fluorescent Protein (RFP) and others,dansyl, umbelliferone. In such conjugates, the antibodies/bindingproteins of the invention can be prepared by methods known to a personskilled in the art. They can then be bound with enzymes or fluorescentlabels directly; via a spacer group or a linkage group such aspolyaldehyde, glutaraldehyde, ethylenediaminetetraacetic acid (EDTA) ordiethylenetriaminepentaacetic acid (DPTA); or in the presence of otherbinding agents such as those routinely known in the art. Conjugatescarrying fluorescein labels can be prepared by, for example, reactionwith an isothiocyanate. In certain situations, the label or marker canalso be therapeutic.

Others conjugates can include chemiluminescent labels such as luminoland dioxetane, bioluminescent labels such as luciferase and luciferin,or radioactive labels such as iodine¹²³, iodine¹²⁵, iodine¹²⁶,iodine^(133, 131), bromine⁷⁷, technetium^(99m), indium¹¹¹,indium^(113m), gallium⁶⁷, gallium⁶⁸, ruthenium⁹⁵, ruthenium⁹⁷,ruthenium¹⁰³, ruthenium¹⁰⁶, mercury¹⁰⁷, mercury²⁰³, rhenium^(99m),rhenium¹⁰¹, rhenium¹⁰⁵, scandium⁴⁷, tellurium^(121m), tellurium^(122m),tellurium^(126m), thulium¹⁶⁵, thulium¹⁶⁷, thulium¹⁶⁸, fluorine¹⁸,yttrium¹⁹⁹ and iodine¹³¹. Existing methods known to a person skilled inthe art for labeling antibodies with radioisotypes, either directly orvia a chelating agent such as the EDTA or DTPA mentioned above, can beused for as diagnostic radioisotopes. See, e.g., labeling with [I¹²⁵]Naby the chloramine-T technique [Hunter W. M. and Greenwood F. C. (1962)Nature 194:495]; labeling with technetium^(99m) as described byCrockford et al. (U.S. Pat. No. 4,424,200); and bound via DTPA asdescribed by Hnatowich (U.S. Pat. No. 4,479,930).

The invention also provides antibodies and other tau-binding moleculesthat can also be used in a method for the diagnosis of a disorder in anindividual by obtaining a body fluid sample from the individual, whichcan be a blood sample, a lymph sample or any other body fluid sample andcontacting the body fluid sample with an antibody of the instantinvention under conditions enabling the formation of antibody-antigencomplexes. The presence and/or amount of such complexes is thendetermined by methods known in the art, a level significantly higherthan that formed in a control sample indicating the presence of diseasein the tested individual. Thus, the present invention relates to an invitro immunoassay comprising an antibody of the invention.

Furthermore, the present invention relates to in vivo imaging techniquesemploying any one of the tau-binding molecules of the present invention.For example, the medical imaging technique Positron emission tomography(PET) which produces a three-dimensional image of body parts is based onthe detection of radiation from the emission of positrons. Typically, abiomolecule is radioactively labeled, e.g. it incorporates a radioactivetracer isotope. Upon administration of the labeled biomolecule to thesubject, typically by injection into the blood circulation, theradioactively labeled biomolecule becomes concentrated in tissues ofinterest. The subject is then placed in the imaging scanner, whichdetects the emission of positrons. In one embodiment, a labeled, forexample ⁶⁴Cu labeled binding molecule such as an antibody isadministered to a subject and detection of the binding molecule and thusmisdisordered or misordered tau is performed by placing the subject inan imaging scanner and detecting the emission of positrons, therebyindicating a neurological disorder if emission is detected. The presentinvention thus encompasses a method for PET imagining, comprising thestep of administering a ⁶⁴Cu-labelled or equivalent labeled bindingmolecule of the present invention to a subject.

The present invention also provides an article of manufacture, such aspharmaceutical and diagnostic packs or kits comprising one or morecontainers filled with one or more of the above described ingredients,i.e. binding molecule, antibody or binding fragment thereof,polynucleotide, vector or cell, as provided by the invention. Associatedwith such container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition or alternatively the kit comprises reagents and/or instructionsfor use in appropriate diagnostic assays. The composition or kit of thepresent invention is suitable for the diagnosis, prevention, andtreatment of Alzheimer's disease and related tauopathies

The biological activity of the binding molecules, e.g., antibodiesprovided by the invention suggests that they have sufficient affinity tomake them candidates for drug localization/drug delivery to cells ortissue. The targeting and binding to misdisordered tau deposits could beuseful for the delivery of therapeutically or diagnostically activeagents and gene therapy/gene delivery. Thus, the invention provides forthe use of the antibodies described herein for the preparation of acomposition for, and in methods of, detection and/or targeting atherapeutic or diagnostic agent to pathological tau and neurofibrillarylesions of the brain. These compositions and methods can be used as partof a treatment protocol for AD and related tauopathies.

Accordingly, the present invention relates to compositions comprisingone or more of the aforementioned compounds, including bindingmolecules, antibodies, binding fragments; chemical derivatives thereof;polynucleotides, vectors, and cells. Certain compositions can furthercomprise one or more pharmaceutically acceptable carriers and one ormore pharmaceutically acceptable diluents. Certain chemical derivativescomprise chemical moieties that are not normally a part of the basemolecule or cell (e.g., of the antibody, binding molecule,polynucleotides, vectors, and cells) but are linked to them by routinemethods. Such moieties can function to, for example, improve thesolubility, half-life, visualization, detectability, and/or absorption,of the base molecule or cell. Alternatively, the moieties can attenuateundesirable side effects of the base molecule or decrease the toxicityof the base molecule.

The invention also provides pharmaceutical compositions comprisingcombinations of the antibodies provided herein with further agents, suchas with interleukins or interferons, depending on the intended use ofthe pharmaceutical composition. For example, for use in the treatment ofAlzheimer's disease the additional agent can be selected from the groupconsisting of small organic molecules, anti-tau antibodies,anti-beta-amlyoid antibodies, and combinations thereof. Other agentsinclude, but are not limited to, acetylcholinesterase inhibitors, NMDAreceptor antagonists, transition metal chelators, growth factors,hormones, non-steroidal anti-inflammatory drugs (NSAID), antioxidants,lipid lowering agents, selective phosphodiesterase inhibitors,inhibitors of tau aggregation, inhibitors of protein kinases, inhibitorsof heat shock proteins, anti-amyloid-passive and -active immunizationreagents, anti-amyloid aggregation inhibitors, and secretase inhibitors.Hence, in an embodiment, the present invention relates to the use of thebinding molecule, antibody or binding fragment of the present inventionor of a binding molecule having substantially the same bindingspecificities of any one thereof, the polynucleotide, the vector or thecell of the present invention for the preparation of a pharmaceutical ordiagnostic composition for treating or preventing the progression ofAlzheimer's disease or related tauopathies; for the amelioration ofsymptoms associated with Alzheimer's disease or related tauopathies; fordiagnosing or screening a subject for the presence of Alzheimer'sdisease or related tauopathies for determining a subject's risk fordeveloping Alzheimer's disease or related tauopathies.

Peptides for Diagnostics, Active Immunization, and AD-Therapy

The present invention is based in part on the discovery that certainfragments of tau are active in inducing an immune response topathological tau when injected into rat models of AD, and would beexpected to do so in humans. These immunogenic tau fragments, whichcomprise one or more of the regions of tau identified above, throughDC8E8, as promoters or at least participants in the development andprogression of AD, were found capable of (i) promoting clearance ofextracellular tau deposits within AD brains (rat models); (ii) inducingthe production of protective antibodies against AD in an animal model;and/or (iii) slowing the progression of AD in the recipient subjects, asmeasured by one or more biochemical and neurological assays, in ananimal model. They can also directly physically interfere with theability of tau to form pathological tau-tau interactions along theseregions

The invention provides immunogens or immunogenic peptides derived fromnewly identified regions of the tau protein that are important for theformation of the core of PHFs and promote PHF assembly in vitro.Strategically targeting these regions (“therapeutic epitopes”) can leadto the successful treatment of AD and related tauopathies. Immunogenscan be screened for therapeutic efficacy in an animal model, such as thetransgenic rat models described below.

In an embodiment of the present invention, tau peptides for exampleencompass one of the following amino acid sequences, within which isseparetely comprised each of the four therapeutic epitopes: a) SEQ IDNO:98 tau 267-KHQPGGG-273, b) SEQ ID NO:99 tau 298-KHVPGGG-304, c) SEQID NO:100 tau 329-HHKPGGG-335, and d) SEQ ID NO:101 tau 361-THVPGGG-367(numbered according to the longest human tau isoform tau 2N4R, 441residues-long, see SEQ ID NO:102). In another embodiment, tau peptidescomprise at least one therapeutic epitope, wherein the therapeuticepitope is selected from SEQ ID NO:223 tau 268-HQPGGG-273, SEQ ID NO:154tau 299-HVPGGG-304, SEQ ID NO:224 tau 330-HKPGGG-335, and SEQ ID NO:154tau 362-HVPGGG-367.

The invention provides 30-amino acid long immunogens, such as any one ofthe SEQ ID NOs shown in the Table 1. Each one of the immunogens includedin Table 1 is an isolated fragment of tau that contains one of thetherapeutic epitopes, located within SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO:100, and SEQ ID NO:101.

TABLE 1 Tau 30-mer peptides each carrying one therapeutic epitopeSEQ ID NO Immunogen Sequences SEQ ID NO: 1 Tau251-280PDLKNVKSKIGS TENLKHQPGGGKVQIINK SEQ ID NO: 2 Tau256-285VKSKIGSTENLKHQP GGGKVQIINK KLDLS SEQ ID NO: 3 Tau259-288KIGSTENLKHQP GGGKVQIINK KLDLSNVQ SEQ ID NO: 4 Tau275-304VQIINKKLDL SNVQSKCGSK DNIKHVPGGG SEQ ID NO: 9 Tau244-273QTAPVPMPDLKNVKSKIGSTENLKHQPGGG SEQ ID NO: 10 Tau245-274TAPVPMPDLKNVKSKIGSTENLKHQPGGGK SEQ ID NO: 11 Tau246-275APVPMPDLKNVKSKIGSTENLKHQPGGGKV SEQ ID NO: 12 Tau247-276PVPMPDLKNVKSKIGSTENLKHQPGGGKVQ SEQ ID NO: 13 Tau248-277VPMPDLKNVKSKIGSTENLKHQPGGGKVQI SEQ ID NO: 14 Tau249-278PMPDLKNVKSKIGSTENLKHQPGGGKVQII SEQ ID NO: 15 Tau250-279MPDLKNVKSKIGSTENLKHQPGGGKVQIIN SEQ ID NO: 16 Tau252-281DLKNVKSKIGSTENLKHQPGGGKVQIINKK SEQ ID NO: 17 Tau253-282LKNVKSKIGSTENLKHQPGGGKVQIINKKL SEQ ID NO: 18 Tau254-283KNVKSKIGSTENLKHQPGGGKVQIINKKLD SEQ ID NO: 19 Tau255-284NVKSKIGSTENLKHQPGGGKVQIINKKLDL SEQ ID NO: 20 Tau257-286KSKIGSTENLKHQPGGGKVQIINKKLDLSN SEQ ID NO: 21 Tau258-287SKIGSTENLKHQPGGGKVQIINKKLDLSNV SEQ ID NO: 22 Tau260-289IGSTENLKHQPGGGKVQIINKKLDLSNVQS SEQ ID NO: 23 Tau261-290GSTENLKHQPGGGKVQIINKKLDLSNVQSK SEQ ID NO: 24 Tau262-291STENLKHQPGGGKVQIINKKLDLSNVQSKC SEQ ID NO: 25 Tau263-292TENLKHQPGGGKVQIINKKLDLSNVQSKCG SEQ ID NO: 26 Tau264-293ENLKHQPGGGKVQIINKKLDLSNVQSKCGS SEQ ID NO: 27 Tau265-294NLKHQPGGGKVQIINKKLDLSNVQSKCGSK SEQ ID NO: 28 Tau266-295LKHQPGGGKVQIINKKLDLSNVQSKCGSKD SEQ ID NO: 29 Tau267-296KHQPGGGKVQIINKKLDLSNVQSKCGSKDN SEQ ID NO: 30 Tau276-305QIINKKLDLSNVQSKCGSKDNIKHVPGGGS SEQ ID NO: 31 Tau277-306IINKKLDLSNVQSKCGSKDNIKHVPGGGSV SEQ ID NO: 32 Tau278-307INKKLDLSNVQSKCGSKDNIKHVPGGGSVQ SEQ ID NO: 33 Tau279-308NKKLDLSNVQSKCGSKDNIKHVPGGGSVQI SEQ ID NO: 34 Tau280-309KKLDLSNVQSKCGSKDNIKHVPGGGSVQIV SEQ ID NO: 35 Tau281-310KLDLSNVQSKCGSKDNIKHVPGGGSVQIVY SEQ ID NO: 36 Tau282-311LDLSNVQSKCGSKDNIKHVPGGGSVQIVYK SEQ ID NO: 37 Tau283-312DLSNVQSKCGSKDNIKHVPGGGSVQIVYKP SEQ ID NO: 38 Tau284-313LSNVQSKCGSKDNIKHVPGGGSVQIVYKPV SEQ ID NO: 39 Tau285-314SNVQSKCGSKDNIKHVPGGGSVQIVYKPVD SEQ ID NO: 40 Tau286-315NVQSKCGSKDNIKHVPGGGSVQIVYKPVDL SEQ ID NO: 41 Tau287-316VQSKCGSKDNIKHVPGGGSVQIVYKPVDLS SEQ ID NO: 42 Tau288-317QSKCGSKDNIKHVPGGGSVQIVYKPVDLSK SEQ ID NO: 43 Tau289-318SKCGSKDNIKHVPGGGSVQIVYKPVDLSKV SEQ ID NO: 44 Tau290-319KCGSKDNIKHVPGGGSVQIVYKPVDLSKVT SEQ ID NO: 45 Tau292-321GSKDNIKHVPGGGSVQIVYKPVDLSKVTSK SEQ ID NO: 46 Tau293-322SKDNIKHVPGGGSVQIVYKPVDLSKVTSKC SEQ ID NO: 47 Tau294-323KDNIKHVPGGGSVQIVYKPVDLSKVTSKCG SEQ ID NO: 48 Tau295-324DNIKHVPGGGSVQIVYKPVDLSKVTSKCGS SEQ ID NO: 49 Tau296-325NIKHVPGGGSVQIVYKPVDLSKVTSKCGSL SEQ ID NO: 50 Tau297-326IKHVPGGGSVQIVYKPVDLSKVTSKCGSLG SEQ ID NO: 51 Tau298-327KHVPGGGSVQIVYKPVDLSKVTSKCGSLGN SEQ ID NO: 52 Tau307-336QIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ SEQ ID NO: 53 Tau308-337IVYKPVDLSKVTSKCGSLGNIHHKPGGGQV SEQ ID NO: 54 Tau309-338VYKPVDLSKVTSKCGSLGNIHHKPGGGQVE SEQ ID NO: 55 Tau310-339YKPVDLSKVTSKCGSLGNIHHKPGGGQVEV SEQ ID NO: 56 Tau311-340KPVDLSKVTSKCGSLGNIHHKPGGGQVEVK SEQ ID NO: 57 Tau312-341PVDLSKVTSKCGSLGNIHHKPGGGQVEVKS SEQ ID NO: 58 Tau313-342VDLSKVTSKCGSLGNIHHKPGGGQVEVKSE SEQ ID NO: 59 Tau314-343DLSKVTSKCGSLGNIHHKPGGGQVEVKSEK SEQ ID NO: 60 Tau315-344LSKVTSKCGSLGNIHHKPGGGQVEVKSEKL SEQ ID NO: 61 Tau316-345SKVTSKCGSLGNIHHKPGGGQVEVKSEKLD SEQ ID NO: 62 Tau317-346KVTSKCGSLGNIHHKPGGGQVEVKSEKLDF SEQ ID NO: 63 Tau318-347VTSKCGSLGNIHHKPGGGQVEVKSEKLDFK SEQ ID NO: 64 Tau319-348TSKCGSLGNIHHKPGGGQVEVKSEKLDFKD SEQ ID NO: 65 Tau320-349SKCGSLGNIHHKPGGGQVEVKSEKLDFKDR SEQ ID NO: 66 Tau321-350KCGSLGNIHHKPGGGQVEVKSEKLDFKDRV SEQ ID NO: 67 Tau322-351CGSLGNIHHKPGGGQVEVKSEKLDFKDRVQ SEQ ID NO: 68 Tau323-352GSLGNIHHKPGGGQVEVKSEKLDFKDRVQS SEQ ID NO: 69 Tau324-353SLGNIHHKPGGGQVEVKSEKLDFKDRVQSK SEQ ID NO: 70 Tau325-354LGNIHHKPGGGQVEVKSEKLDFKDRVQSKI SEQ ID NO: 71 Tau326-355GNIHHKPGGGQVEVKSEKLDFKDRVQSKIG SEQ ID NO: 72 Tau327-356NIHHKPGGGQVEVKSEKLDFKDRVQSKIGS SEQ ID NO: 73 Tau328-357IHHKPGGGQVEVKSEKLDFKDRVQSKIGSL SEQ ID NO: 74 Tau329-358HHKPGGGQVEVKSEKLDFKDRVQSKIGSLD SEQ ID NO: 75 Tau339-368VKSEKLDFKDRVQSKIGSLDNITHVPGGGN SEQ ID NO: 76 Tau340-369KSEKLDFKDRVQSKIGSLDNITHVPGGGNK SEQ ID NO: 77 Tau341-370SEKLDFKDRVQSKIGSLDNITHVPGGGNKK SEQ ID NO: 78 Tau342-371EKLDFKDRVQSKIGSLDNITHVPGGGNKKI SEQ ID NO: 79 Tau343-372KLDFKDRVQSKIGSLDNITHVPGGGNKKIE SEQ ID NO: 80 Tau344-373LDFKDRVQSKIGSLDNITHVPGGGNKKIET SEQ ID NO: 81 Tau345-374DFKDRVQSKIGSLDNITHVPGGGNKKIETH SEQ ID NO: 82 Tau346-375FKDRVQSKIGSLDNITHVPGGGNKKIETHK SEQ ID NO: 83 Tau347-376KDRVQSKIGSLDNITHVPGGGNKKIETHKL SEQ ID NO: 84 Tau348-377DRVQSKIGSLDNITHVPGGGNKKIETHKLT SEQ ID NO: 85 Tau349-378RVQSKIGSLDNITHVPGGGNKKIETHKLTF SEQ ID NO: 86 Tau350-379VQSKIGSLDNITHVPGGGNKKIETHKLTFR SEQ ID NO: 87 Tau351-380QSKIGSLDNITHVPGGGNKKIETHKLTFRE SEQ ID NO: 110 Tau352-381SKIGSLDNITHVPGGGNKKIETHKLTFREN SEQ ID NO: 89 Tau353-382KIGSLDNITHVPGGGNKKIETHKLTFRENA SEQ ID NO: 90 Tau354-383IGSLDNITHVPGGGNKKIETHKLTFRENAK SEQ ID NO: 91 Tau355-384GSLDNITHVPGGGNKKIETHKLTFRENAKA SEQ ID NO: 92 Tau356-385SLDNITHVPGGGNKKIETHKLTFRENAKAK SEQ ID NO: 93 Tau357-386LDNITHVPGGGNKKIETHKLTFRENAKAKT SEQ ID NO: 94 Tau358-387DNITHVPGGGNKKIETHKLTFRENAKAKTD SEQ ID NO: 95 Tau359-388NITHVPGGGNKKIETHKLTFRENAKAKTDH SEQ ID NO: 96 Tau360-389ITHVPGGGNKKIETHKLTFRENAKAKTDHG SEQ ID NO: 97 Tau361-390THVPGGGNKKIETHKLTFRENAKAKTDHGA

In some embodiments, the immunogenic peptide is chosen from SEQ ID NO:1tau 251-PDLKNVKSKIGSTENLKHQPGGGKVQIINK-280; SEQ ID NO:2 tau256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285; SEQ ID NO:3 tau259-KIGSTENLKHQPGGGKVQIINK KLDLSNVQ-288; and SEQ ID NO:4 tau275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304.

The invention also provides for shorter and longer immunogenic peptidesfor use in the present invention that contain one or more of the aminoacid sequences SEQ ID NO:98 267-KHQPGGG-273, or amino acids SEQ ID NO:99298-KHVPGGG-304, or amino acids SEQ ID NO:100 329-HHKPGGG-335, or aminoacids SEQ ID NO:101 361-THVPGGG-367 can be derived from any one of thesix isoforms of human tau protein. In one embodiment, an immunogenicpeptide comprises at least one therapeutic epitope, wherein thetherapeutic epitope is selected from SEQ ID NO:223 tau 268-HQPGGG-273,SEQ ID NO:154 tau 299-HVPGGG-304, SEQ ID NO:224 tau 330-HKPGGG-335, andSEQ ID NO:154 tau 362-HVPGGG-367. In one embodiment, the immunogenicpeptide comprises a sequence selected from SEQ ID NO:109 Tau314-DLSKVTSKCGSLGNIHHKPGGGQVEVKSE-342; SEQ ID NO:110 Tau352-SKIGSLDNITHVPGGGNKKIETHKLTFREN-380; SEQ ID NO:111 Tau325-LGNIHHKPGGGQ-336; SEQ ID NO:112 Tau 357-LDNITHVPGGGN-368; SEQ IDNO:108 Tau 294-305 KDNIKHVPGGGS. In some embodiments, at least oneimmunogenic peptide is chosen from any one of SEQ ID NOs: 1-4, SEQ IDNOs: 9-101, and SEQ ID NOs: 108-112, NIKAVPGGGS (SEQ ID NO: 200),NIKHVPGGGS (SEQ ID NO: 201), IKHVPGGGS (SEQ ID NO: 202), KHVPGGGSV (SEQID NO: 203), HVPGGGSVQ (SEQ ID NO: 204), VPGGGSVQ (SEQ ID NO: 205),GWSIHSPGGGSC (SEQ ID NO: 250), and SVFQHLPGGGSC (SEQ ID NO: 251),ANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS (SEQ ID NO: 146), DNAKHVPGGGS(SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO: 151), DNIKAVPGGGS (SEQ ID NO:159), DNIKHAPGGGS (SEQ ID NO: 161), and DNIKHVPGGGS (SEQ ID NO: 171).

The amino acid sequences corresponding to the human tau isoforms aregiven in SEQ ID NOs:102-107

SEQ ID NO: 102 (2N4R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLVDEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAGHVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPPGQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSPGSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAKSRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINKKLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVDLSKVTSKCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNITHVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVSGDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L SEQ ID NO: 103 (1N4R):MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEAEEAGIGDTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKTKIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPPKSGDRSGYSS PGSPGTPGSR SRTPSLPTPP TREPKKVAVVRTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQPGGGKVQIIN KKLDLSNVQS KCGSKDNIKH VPGGGSVQIVYKPVDLSKVT SKCGSLGNIH HKPGGGQVEV KSEKLDFKDRVQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHGAEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GLSEQ ID NO: 104 (2N3R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLVDEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAGHVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPPGQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSPGSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAKSRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIVYKPVDLSKVTSK CGSLGNIHHK PGGGQVEVKS EKLDFKDRVQSKIGSLDNIT HVPGGGNKKI ETHKLTFREN AKAKTDHGAEIVYKSPVVSG DTSPRHLSNV SSTGSIDMVD SPQLATLADE VSASLAKQGLSEQ ID NO: 105 (0N4R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKAEEAGI GDTPSLEDEA AGHVTQARMV SKSKDGTGSDDKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPAPKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTPPTREPKKVAV VRTPPKSPSS AKSRLQTAPV PMPDLKNVKSKIGSTENLKH QPGGGKVQII NKKLDLSNVQ SKCGSKDNIKHVPGGGSVQI VYKPVDLSKV TSKCGSLGNI HHKPGGGQVEVKSEKLDFKD RVQSKIGSLD NITHVPGGGN KKIETHKLTFRENAKAKTDH GAEIVYKSPV VSGDTSPRHL SNVSSTGSID MVDSPQLATL ADEVSASLAK QGLSEQ ID NO: 106 (1N3R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEAEEAGIGDTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKTKIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPPKSGDRSGYSS PGSPGTPGSR SRTPSLPTPP TREPKKVAVVRTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQPGGGKVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVKSEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRENAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG LSEQ ID NO: 107 (0N3R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKAEEAGI GDTPSLEDEA AGHVTQARMV SKSKDGTGSDDKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPAPKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTPPTREPKKVAV VRTPPKSPSS AKSRLQTAPV PMPDLKNVKSKIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIHHKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNKKIETHKLTFR ENAKAKTDHG AEIVYKSPVV SGDTSPRHLSNVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL

The use of peptide-based vaccines to elicit immune responses in diseasesfor which no conventional vaccines are yet available is attractive(Brown, 1994; BenYedidia, et al., 1997). However, in many cases smallpeptides are poor immunogens because they act as haptens that lack thenecessary Th-cell epitopes and/or that are captured with low efficiencyby antigen presenting cells (APC). In one embodiment of the presentinvention, the immunogenic epitopes can be longer polypeptides thatinclude a protective epitope of tau peptide, or analogue together withother amino acids.

Some of the agents described herein for inducing an immune responsecontain the appropriate epitope for inducing an immune response againstpathological tau and tau deposits but are too small to be immunogenic.In this case, a peptide immunogen can be linked to a suitable carrier tohelp elicit an immune response. In certain embodiments, suitablecarriers include serum albumins, keyhole limpet hemocyanin,immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or atoxoid from other pathogenic bacteria, such as diphtheria, E. coli,cholera, or H. pylori, or an attenuated toxin derivative. Other carriersfor stimulating or enhancing an immune response include cytokines suchas IL-1, IL-1α and β peptides, IL-2, γINF, IL-10, GM-CSF, andchemokines, such as M1P1α and β and RANTES. Immunogenic agents can alsobe linked to peptides that enhance transport across tissues, asdescribed in O'Mahony, WO 97/17613 and WO 97/17614.

Immunogenic agents can be linked to carriers by chemical crosslinking.Techniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) (if the peptide lacks a sulfhydryl group, this can be provided byaddition of a cysteine residue). These reagents create a disulfidelinkage between themselves and peptide cysteine resides on one proteinand an amide linkage through the .epsilon.-amino on a lysine, or otherfree amino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenic peptides can also be expressed as fusion proteins withcarriers. The immunogenic peptide can be linked at the amino terminus,the carboxyl terminus, or at a site anywhere within the peptide(internally) to the carrier. In some embodiments, multiple repeats ofthe immunogenic peptide can be present in the fusion protein.

For example, also provided are immunogens that include fusion proteinscomprising a tau peptide carrying a protective B cell epitope linked toa promiscuous non-natural Pan DR Th-cell epitope that induces a B-cellresponse against the protective epitope. In a further alternative, theinvention provides immunogens that can be designed as polymers (Jacksonet a., 1997), multiple antigen peptide systems (MAP) (Tam and Recent,1996), immunostimulating complexes (ISCOM) (Barr, I. G. and Mitchell,1996) and possibly other branched amphoteric polypeptides (Wilkinson etal., 1998), or chimeric peptides produced by co-linearization of theepitopes (Marussig et al., 1997).

In certain embodiments, the therapeutic peptides can be applied alone orin combination, bound or not to a pharmaceutically acceptable carrierincluding KLH, tetanus toxoid, albumin binding protein, bovine serumalbumin, dendrimer (MAP; Biol. Chem. 358: 581) as well as adjuvantsubstances, or their combinations, described e.g. in O'Hagan et al.(2003) (in particular the endogenous immunopotentiating compounds anddispensing systems described therein) and in Wilson-Welderer et al.(2009) (in particular those indicated in table 2 and 3 of said document)or mixtures thereof.

In certain embodiments, an immunogenic agent of the present inventioncan be bound or linked to a suitable carrier by chemical crosslinking toincrease the immune response against pathological tau, including taudeposits. In certain embodiments, the bound or linked pharmaceuticallyacceptable carrier is keyhole limpet hemocyanin (KLH), tetanus toxoid,bovine serum albumin (BSA), immunoglobulin (Ig) molecule, thyroglobulin,or ovoglobulin. Other carriers for stimulation of immune responseinclude cytokines (such as IL-1, IL-2, IL-10 IFNγ, GM-CSF) andchemokines (such as M1P1α and β).

Tau peptides or analogs can be synthesized by solid phase peptidesynthesis or recombinant expression, or can be obtained from naturalsources. Automatic peptide synthesizers are commercially available fromnumerous suppliers, such as Applied Biosystems, EZBiolab, or Antagene.Recombinant expression systems can include bacteria, such as E. coli,yeast, insect cells, or mammalian cells. Procedures for the manipulationof DNA and preparation DNA constructs for recombinant expression aredescribed by Sambrook et al. (1989), methods for the production ofrecombinant proteins are described in detail in Current Protocols inProtein Science (Chapter 5 “Production of Recombinant Proteins”, UNITS5.1-5.24, DOI: 10.1002/0471140864, also available online atonlinelibrary.wiley.com/book/10.1002/0471140864/toc).

The immunogenic agents of the present invention can be expressed by avirus or bacteria as a vector or carrier. A nucleic acid encoding theimmunogenic peptide is incorporated into a genome or episome of thevirus or bacteria. Finally, the immunogenic peptides can be expressed asa secreted protein or as a fusion protein with an outer surface proteinof a virus or can be displayed as a transmembrane protein of bacteria.Viruses or bacteria used in such methods are generally nonpathogenic orattenuated. Suitable viruses include adenovirus, HSV, Venezuelan equineencephalitis virus and other alpha viruses, vesicular stomatitis virus,and other rhabdo viruses, vaccinia and fowl pox. Suitable bacteriainclude Salmonella and Shigella. Alternatively, fusion of an immunogenicpeptide to HBsAg of HBV is suitable.

A further aspect of the present invention relates to the therapeuticagent or immunogen, which can also be an analogue of the variouspeptides described in the various embodiments (e.g., SEQ ID No: 1-4;9-97) or of fragments thereof.

The present invention is also based on the discovery of novel peptides,designated in this application as designer therapeutic epitopes. Despitehaving a primary sequence that is different from that of tau and taufragments, this invention features designer therapeutic epitopes thatare capable of having a shape (e.g., an intrinsically disorderedstructure, a tertiary structure, a conformation) that mimics that of oneor more of the tau “therapeutic epitopes” described above. By mimickingone or more of these regions, these designer therapeutic epitopes can beuseful to generate antibodies against them, such as antibodies thatcompete with DC8E8. These peptides are able to compete with tau or taufragments for binding to the DC8E8 antibody, disclosed above.

Also included are immunogenic designer therapeutic epitopes capable ofinducing an immune response to pathological tau when injected into ratmodels of AD and which would be expected to do so in humans. Inaddition, also disclosed are mouse antibodies/antisera, produced inresponse to immunization with one or more designer therapeutic epitopes,and capable of (i) recognizing one or more epitopes that are or mimicthose of DC8E8; (ii) discriminating between pathological tau and normaltau; and/or (iii) recognizing neurofibrillary lesions in human AD brainand/or in transgenic rat models of AD.

The invention also provides compositions for the prevention, treatment,and/or diagnosis of Alzheimer's disease, wherein the compositioncomprises (i) a means for treating Alzheimer's disease in a subject byinhibiting tau-tau aggregation; and (2) a pharmaceutically acceptablecarrier and/or diluent. The invention also provides compositions for theprevention, treatment, and/or diagnosis of Alzheimer's disease, whereinthe composition comprises (i) a means for treating Alzheimer's diseasein a subject by binding to one or more “therapeutic epitopes” inpathological tau; and (2) a pharmaceutically acceptable carrier and/ordiluents. The invention also provides compositions for the prevention,treatment, and/or diagnosis of Alzheimer's disease, wherein thecomposition comprises (i) a means for decreasing tau-tau aggregation bybinding to one or more “therapeutic epitopes” in pathological tau; and(2) a pharmaceutically acceptable carrier and/or diluents.

Formulations

Agents of the invention can be administered as pharmaceuticalformulations comprising a therapeutic agent (e.g., antibody or peptide,as described above) and one or more of other pharmaceutically acceptablecomponents. See Remington's Pharmaceutical Science (15th ed., MackPublishing Company, Easton, Pa., 1980). These formulations include, forexample, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as LIPOFECTIN), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures can be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration. See also Baldrick P.“Pharmaceutical excipient development: the need for preclinicalguidance.” Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W.“Lyophilization and development of solid protein pharmaceuticals.”Int.J. Pharm. 203(1-2):1-60 (2000), Charman W N “Lipids, lipophilic drugs,and oral drug delivery-some emerging concepts.” J. Pharm Sci.89(8):967-78 (2000), Powell et al. “Compendium of excipients forparenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) andthe citations therein for additional information related toformulations, excipients and carriers known to pharmaceutical chemists.

A wide variety of pharmaceutically acceptable excipients are known inthe art and need not be discussed in detail herein. Pharmaceuticallyacceptable excipients have been amply described in a variety ofpublications, including, for example, A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy,” 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7.sup.th ed., Lippincott, Williams, & Wilkins;and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al.,eds., 3 ed. Amer. Pharmaceutical Assoc.

The chosen formulation depends on the intended mode of administrationand therapeutic application. The formulations can also includepharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Thepharmaceutical composition or formulation can also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like. However, some reagents suitable foradministration to animals, such as Complete Freund's adjuvant are nottypically included in compositions for human use.

Examples of suitable pharmaceutical carriers are known in the art andinclude phosphate buffered saline solutions, water, emulsions, such asoil/water emulsions, various types of wetting agents, sterile solutionsetc. Compositions comprising such carriers can be formulated by knownconventional methods. More carriers are described further below.

Adjuvants

Therapeutic agents, immunogens, of the invention can be administered incombination with adjuvants, i.e., substances that do not themselvescause adaptive immune responses, but amplify or modulate the response toan accompanying antigen. A variety of adjuvants can be used incombination with the therapeutic peptides and antibodies in the presentinvention, in order to elicit an immune response. Preferred adjuvant(s)augment the intrinsic response to an immunogen without causingconformational changes in the immunogen that would affect thequalitative form of the response.

In certain embodiments, the adjuvant is an aluminum salt (alum), such asaluminum hydroxide, aluminum phosphate, and aluminum sulphate (Hunter,2002). Such adjuvants can be used with or without other specificimmunostimulating agents, such as 3 de-O-acylated monophosphoryl lipid A(MPL) or 3-DMP, polymeric or monomeric amino acids, such as polyglutamicacid or polylysine. Such adjuvants can be used with or without otherspecific immunostimulating agents, such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wallcomponents. Other adjuvants are oil-in-water emulsions and include (a)MF59 (WO 90/14837 to Van Nest et al., which is hereby incorporated byreference in its entirety), containing 5% Squalene, 0.5% Tween 80, and0.5% Span 85 (optionally containing various amounts of MTP-PE)formulated into submicron particles using a microfluidizer such as Model110Y microfuidizer (Microfluidics, Newton Mass.), (b) SAF, containing10% Squalene, 0.4% Tween 80, 5% pluronic-bloeked polymer L121, andthr-MDP, either microfluidized into a submicron emulsion or vortexed togenerate a larger particle size emulsion, and (c) Ribi™ adjuvant system(RAS), (Ribi InunoChem, Hamilton, Mont.) containing 2% squalene, 0.2%Tween 80, and one or more bacterial cell wall components from the groupconsisting of monophosphoryllipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), for example MPL-FCWS (Detox™). In someembodiments, the adjuvant isa saponin, such as Stimulon™ (QS21, Aquila,Worcester, Mass.) or particles generated therefrom such as ISCOMs(immunostimulating complexes) and ISCOMATRIX. Other adjuvants includeComplete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA),cytokines, such as interleukins (IL-1, IL-2, and IL-12), macrophagecolony stimulating factor (M-CSF), and tumor necrosis factor (TNF).

Alternatively, tau peptides and their immunogenic analogues can becoupled to an adjuvant. For example, a lipopeptide version of a taupeptide “A” can be prepared by coupling palmitic acid or other lipidsdirectly to the N-terminus of “A” as described for hepatitis B antigenvaccination (Livingston, J. Immunol. 159, 1383-1392 (1997)). However,such coupling should not substantially change the conformation of thetau peptide “A” so as to affect the nature of the immune responsethereto.

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with, or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel, indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on the stability of the immunogenic formulationcontaining the adjuvant, the route of administration, the dosingschedule, the efficacy of the adjuvant for the species being vaccinated,and, in humans, a pharmaceutically acceptable adjuvant is one that hasbeen approved or is approvable for human administration by pertinentregulatory bodies. For example, Complete Freund's adjuvant is notsuitable for human administration. However, alum, MPL or IncompleteFreund's adjuvant (Chang et al., Advanced Drug Delivery Reviews32:173-186 (1998), which is hereby incorporated by reference in itsentirety) alone or optionally in combination with any of alum, QS21, andMPL and all combinations thereof are suitable for human administration.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules, such as proteins, polysaccharides like chitosan,polylactic acids, polyglycolic acids and copolymers (e.g., latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (e.g., oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

Combinations

The invention provides for compositions and methods of treatment thatcombine the antibodies and peptides described herein with othertreatments for AD and related tauopathies. For example, currently,tau-related therapeutic strategies mainly focus on the drugs thatinhibit tau kinases or activate phosphatases (Iqbal and Grundke-lqbal,2004, 2005, 2007; Noble et al. 2005), the drugs that stabilizemicrotubules (Zhang et al. 2005), the drugs that facilitate theproteolytic degradation of misfolded tau protein (Dickey et al. 2005;Dickey and Petrucelli, 2006; Dickey et al. 2006), compounds that preventor reverse tau aggregation (Wischik et al. 1996; Pickhardt et al. 2005;Taniguchi et al. 2005; Necula et al. 2005; Larbig et al. 2007) orvaccine-mediated clearance of aggregated tau (Asuni et al. 2007).Therefore, the invention provides that multiple targeting (e.g.,targeting both tau and beta-amyloid) can substantially increasetreatment efficiency.

In the case of Alzheimer's disease and related tauopathies, in whichpathological soluble tau and insoluble tau (tau deposits) occur in thebrain, agents of the invention can also be administered in conjunctionwith other agents that increase passage of the agents of the inventionacross the blood-brain barrier.

Methods of Administration

Agents for inducing an immune response (passive or active), for reducingthe level of tau, or for any of the methods of prevention, treatment, ordiagnosis (in vivo) described herein, can be administered by parenteral,topical, intradermal, intravenous, oral, subcutaneous, intraperitoneal,intranasal or intramuscular means for prophylactic and/or therapeutictreatment. A typical route of administration is subcutaneous althoughothers can be equally effective. Another typical route is intramuscularinjection. This type of injection is most typically performed in the armor leg muscles. Intravenous injections as well as intraperitonealinjections, intraarterial, intracranial, or intradermal injections arealso effective in generating an immune response. In some methods, agentsare injected directly into a particular tissue where deposits haveaccumulated.

Aerosol formulations such as nasal spray formulations include purifiedaqueous or other solutions of the active agent with preservative agentsand isotonic agents. Such formulations are for example adjusted to a pHand isotonic state compatible with the nasal mucous membranes.Formulations for rectal or vaginal administration can be presented as asuppository with a suitable carrier.

For parenteral administration, therapeutic peptides of the presentinvention can be administered as injectable dosages of a solution orsuspension of the substance in a physiologically acceptable diluent witha pharmaceutical carrier that can be a sterile liquid such as water,oil, saline, glycerol, or ethanol. Additionally, auxiliary substances,such as wetting or emulsifying agents, surfactants, pH bufferingsubstances and the like can be present in compositions. Other componentsof pharmaceutical compositions are those of petroleum, animal,vegetable, or synthetic origin. Peanut oil, soybean oil, and mineral oilare all examples of useful materials. In general, glycols, such aspropylene glycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions. Agents of the invention can alsobe administered in the form of a depot injection or implant preparationwhich can be formulated in such a manner as to permit a sustainedrelease of the active ingredient. An exemplary composition comprisesmonoclonal antibody at 5 mg/mL, formulated in aqueous buffer consistingof 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).The agents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, for example 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, for example 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin patchor using transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24(1995); Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).Subcutaneous administration of a subject antibody, peptide, or compound,is accomplished using standard methods and devices, e.g., needle andsyringe, a subcutaneous injection port delivery system, and the like.Intramuscular administration is accomplished by standard means, e.g.,needle and syringe, continuous delivery system, etc. In someembodiments, a subject antibody, peptide, and/or compound, is deliveredby a continuous delivery system. The term “continuous delivery system”is used interchangeably herein with “controlled delivery system” andencompasses continuous (e.g., controlled) delivery medical devices(e.g., pumps) in combination with catheters, injection devices, and thelike, a wide variety of which are known in the art. Mechanical orelectromechanical infusion pumps can also be suitable for use with thepresent invention. Examples of such devices include those described in,for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019;4,725,852; 5,820,589; 5,643,207; 6,198,966; and the like. In general,the present methods of drug delivery can be accomplished using any of avariety of refillable, pump systems. Pumps provide consistent,controlled release over time.

The agents can also be administered by the now standard procedure ofdrilling a small hole in the skull to administer a drug. In a preferredaspect, the binding molecule, especially antibody or antibody-based drugof the present invention can cross the blood-brain barrier, which allowsfor intravenous or oral administration.

In pharmaceutical dosage forms, the agents (antibodies, peptides,compounds provided by the invention) can be administered in the form oftheir pharmaceutically acceptable salts, or they can also be used aloneor in appropriate association, as well as in combination, with otherpharmaceutically active compounds. The methods and excipients describedherein are merely exemplary and are in no way limiting. A subjectantibody, peptide, or compound, can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives. Unit dosage forms for injection orintravenous administration can comprise the active agent in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier. The term “unit dosage form,” or“dose,” as used herein, refers to physically discrete units suitable asunitary dosages for human and animal subjects, each unit containing apredetermined quantity of a subject antibody calculated in an amountsufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for the unit dosage forms of the present invention dependon the particular compound employed and the effect to be achieved, andthe pharmacodynamics associated with each compound in the host.

Immune responses against pathological tau proteins and tau deposits canalso be induced by administration of nucleic acids encoding therapeutictau peptides. Such nucleic acids can be DNA or RNA. A nucleic acidsegment encoding the immunogen is linked to regulatory elements, such asa promoter and enhancer that allow expression of such a DNA segment inthe intended target cells of a patient. Usually, promoter and enhancerelements from immunoglobulin genes (light or heavy chain) or the CMVmajor intermediate early promoter and enhancer are suitable to directexpression in the blood cells, which are the desirable target forinduction of an immune response. The linked regulatory elements andcoding sequences are often cloned into a vector.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie et al., Cur. Opin. Genet. Develop. 3:102-109(1993), which is hereby incorporated by reference in its entirety);adenoviral vectors (Bett et al., J. Virol. 67:5911 (1993), which ishereby incorporated by reference in its entirety); adeno-associatedvirus vectors (Zhou et al., J. Exp. Med. 179:1867 (1994), which ishereby incorporated by reference in its entirety), viral vectors fromthe pox family including vaccinia virus and the avian pox viruses, viralvectors from the alpha virus genus, such as those derived from Sindbisand Semliki Forest Viruses (Dubensky et al., J. Virol 70:508-519 (1996),which is hereby incorporated by reference in its entirety), Venezuelanequine encephalitis virus (see U.S. Pat. No. 5,643,576 to Johnston etal., which is hereby incorporated by reference in its entirety) andrhabdoviruses, such as vesicular stomatitis virus (see WO 96/34625 toRose, which is hereby incorporated by reference in its entirety) andpapillomaviruses (Ohe, et al., Human Gene Therapy 6:325-333 (1995); WO94/12629 to Woo et al.; and Xiao & Brandsma, Nucleic Acids. Res.24:2630-2622 (1996), which are hereby incorporated by reference in theirentirety).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed toor associated with particulate carriers, examples of which includepolymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346). DNA can also beadministered using a gene gun (see U.S. Pat. No. 6,436,709). In thisapplication, the DNA encoding an immunogen is precipitated onto thesurface of microscopic metal beads. The microprojectiles are acceleratedwith a shock wave or expanding helium gas, and penetrate tissues to adepth of several cell layers (reviewed in Haynes et al., 1996). Forexample, the Accel™ Gene Delivery Device manufactured by Agacetus, Inc.(Middleton, Wis.) or Helios Gene Gun manufactured by Bio-RadLaboratories, Inc. (Hercules, Calif.) are suitable. For therapeuticpurposes, DNA can also be delivered by electroporation (e.g. asdescribed in Trollet et al., 2008 and references therein).Alternatively, naked DNA can pass through skin into the blood streamsimply by spotting the DNA onto skin with chemical or mechanicalirritation (see WO 95/05853) or tattooing (e.g. as described by van denBerg et al., 2009).

In a different variation, DNA or vectors encoding immunogens can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) oruniversal donor hematopoietic stem cells, followed by reimplantation ofthe cells into a patient, for example after verification of theexpression of the immunogen and usually after selection for cells whichhave incorporated the vector.

Another promising although potentially riskier approach for humantreatment has been to transfect dentritic cells (DCs, through straightDNA delivery or using viral strategies) to produce the antigenthemselves (Xing et al., 2005), thus providing a continuous supply ofintact antigen presented through MHC I.

Subjects Amenable to Treatment

Subjects amenable to treatment include individuals at risk ofAlzheimer's disease or related tauopathies but not showing symptoms, aswell as patients already showing symptoms. In the case of Alzheimer'sdisease, virtually anyone is at risk of suffering from Alzheimer'sdisease if he or she lives long enough. Therefore, the presenttreatments or therapies can even be administered prophylactically to thegeneral population without any assessment of the risk of the subjectpatient. The vaccines presented in this patent can be especially usefulfor individuals who have a known genetic risk of Alzheimer's disease.Such individuals include those having relatives who suffered from thisdisease, and those whose risk is determined by the presence of geneticor biochemical markers. Genetic markers of risk of early onset familialAlzheimer's disease include mutations in the APP gene, presenilin genesPS1 and PS2, and markers for late onset Alzheimer's disease in the ApoE4gene (recently reviewed by Bertram and Tanzi, 2008). Additional riskfactors include family history of AD, hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sdisease can be recognized from characteristic dementia, as well as thepresence of the risk factors described above. In addition, a number ofdiagnostic tests are available for identifying individuals who have AD.These include measurement of total tau, phospho-tau and amyloid 13(1-42) levels in CSF. Elevated tau and/or phospho-tau and decreasedamyloid 13 (1-42) levels indicate the presence of AD. Individualssuffering from Alzheimer's disease can also be diagnosed by MMSE, ADRDAor other criteria.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). However, it may not be necessary to begin treatment until a patientreaches 40, 50, 60 or 70. Treatment can entail multiple dosages over aperiod of time. Treatment can be monitored by assaying antibody, oractivated T-cell or B-cell responses to the therapeutic agent (e.g., taupeptide) over time. If the response falls, a booster dosage isindicated. In the case of potential Down's syndrome patients, who are athigher risk for AD or related tauopathies, treatment can beginpre-natally, by administering therapeutic agent to the mother, orshortly after birth.

In some embodiments, DC8E8 (or a chimeric, humanized, human, or otherderivative/portion/fragment thereof) is the antibody or passive vaccineintended for use in aged immunosenescent Alzheimer's disease (AD)patients showing significant decrease in the levels of theco-stimulatory molecule CD28 on T-cells. A decrease in co-stimulatorymolecule CD28 is indicative of impaired immune response (Saurwein-Teisslet al., 2002). CD8⁺CD28⁻ T cell clones which are frequently CD45RA⁺(immunophenotype: CD8⁺CD28⁻ CD45RA⁺) produce large amount ofpro-inflammatory cytokine IFN-γ and marginal amounts of IL-5. Theseclones accumulate during normal aging and induce imbalance in theproduction of Th₁ and Th₂ cytokines. Thus, the accumulating CD8⁺ CD28⁻CD45RA⁺ T cell clones along with the dwindling population of naïve Bcells (Siegrist and Aspinall, 2009) are the major contributors to thedecline of immune functions that affects about one third of elderlypopulation (Weng et al., 2009, Saurwein-Teissl et al., 2002).

Accordingly, passive immunotherapy (e.g., with DC8E8 (or a chimeric,humanized, human, or other derivative/portion/fragment thereof) providesa means to circumvent the failing immune system of a large population ofAD patients and target the pathological tau proteins causingneurofibrillary degeneration.

In some embodiments, one of the tau therapeutic epitopes (or a peptidecomprising one of the tau therapeutic epitopes described herein) is usedas an active vaccine, intended for use in aged immunocompetentAlzheimer's disease patients. The immunophenotype of immunocompetentpatients is CD8⁺CD28⁺ CD45RA⁺. Therefore levels of co-stimulatorymolecule CD28 on CD8⁺T cells will be determined and used as a selectionmarker of patients for active vaccination.

Furthermore, prior to the treatment, CSF and blood taken from patientswill be tested for antibodies against Borrelia, Treponema, Chlamydia,Herpesvirus and other brain pathogens to exclude individuals withchronic infectious and inflammatory CNS disorders that can mimic oraggravate the symptoms of AD (Balin et al., 2008; Itzhaki and Wozniak,2008; Miklossy, 2008; Andreasen, 2010). CNS infections often compromisethe function of the blood-brain barrier (BBB), especially Chlamydiainfections of the brain endothelial cells, which can can lead to anincreased influx of monocytes into the brain parenchyma and thus caninfluence the local immune response (Balin et al., 2008). It has alsobeen shown that elderly subjects with higher levels of IgG tocytomegalovirus (CMV) suffered faster rates of cognitive decline(Itzhaki and Wozniak, 2008). Therefore, in order to prevent adverseeffects after immunization with one of the agents provided by theinvention (e.g. uncontrolled immune reaction to normal tau) theAlzheimer's disease patients with CNS infections or those testedpositively for antibodies against the aforementioned pathogens will betreated with a highly selective vaccine.

Prior to the treatment, CSF and blood taken from patients can be testedfor antibodies against Borrelia, Treponema, Chlamydia, Herpesvirus andother brain pathogens to exclude individuals with chronic infectious andinflammatory CNS disorders that can mimic or aggravate the symptoms ofAD (Balin et al., 2008; Itzhaki and Wozniak, 2008; Miklossy, 2008;Andreasen, 2010). In order to prevent possible adverse effects promotedby various chronic infections, this group of patients will be treatedwith a more selective antibody or therapeutic-epitope-containingvaccine. In some instances, the active vaccine is a designer epitope(e.g., see EXAMPLES), inducing the production of strictly selectiveantibodies targeting the therapeutic epitope on pathological tauproteins. In some embodiments, the vaccine does not contain any aminoacid sequence shared with normal/physiological tau protein.

Treatment Regimes

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of, aparticular disease in an amount sufficient to eliminate or reduce therisk or delay the outset of the disease. In therapeutic applications,compositions or medicaments are administered to a patient suspected of,or already suffering from such a disease in an amount sufficient tocure, or at least partially arrest, the symptoms of the disease and itscomplications. An amount adequate to accomplish this is defined as atherapeutically or pharmaceutically effective dose. In both prophylacticand therapeutic regimes, agents are usually administered in severaldosages until a sufficient immune response has been achieved. Typically,the immune response is monitored and repeated dosages are given if theimmune response starts to fade.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions, vary depending upon manyfactors, including means of administration, target site, physiologicalstate of the patient, whether the patient is human or an animal, othermedications administered, and whether the treatment is prophylactic ortherapeutic. Usually, the patient is a human. Treatment dosages need tobe titrated to optimize safety and efficacy. Accordingly, treatment withan antibody or tau-binding protein will typically entail multipledosages over a period of time. For passive immunization with anantibody, the dosage ranges from about 0.0001 to 100 mg/kg, and moreusually 0.01 to 5 mg/kg of the host body weight. In some applications,the amount of antibody or tau-binding protein can be administered at adosage of at least 0.1 mg/kg of body weight, at a dosage of at least 0.5mg/kg of body weight, 1 mg/kg of body weight, or any combination ofdosages between 0.1 and 10 mg/kg of body weight. In some methods, theantibody or tau-binding protein can be administered in multiple dosages(equal or different) over a period of at least 1 month, at least 3months, or at least 6 months. The total number of doses over any onetreatment period can be, for example, between 4 and 6, although othernumbers can be used depending on the factors discussed above. Treatmentcan be monitored by any of the methods described further below.

The amount of immunogen depends on whether adjuvant is alsoadministered, with higher dosages being required in the absence ofadjuvant. The amount of an immunogen for administration sometimes variesfrom 1 μg-500 μg per patient and more usually from 5-500 μg perinjection for human administration. Occasionally, a higher dose of 1-2mg per injection is used. Typically about 10, 20, 50 or 100 μg is usedfor each human injection. The timing of injections can varysignificantly from once a day, to once a year, to once a decade. On anygiven day that a dosage of immunogen is given, the dosage is greaterthan 1 μg/patient and usually greater than 10 μg/patient if adjuvant isalso administered, and greater than 10 μg/patient and usually greaterthan 100 μg/patient in the absence of adjuvant. A typical regimenconsists of an immunization followed by booster injections at 6 weeklyintervals. Another regimen consists of an immunization followed bybooster injections 1, 2 and 12 months later. Another regimen entails aninjection every two months for life. Alternatively, booster injectionscan be on an irregular basis as indicated by monitoring of immuneresponse. In some embodiments, the active vaccine will be formulatedwith a suitable carrier, preferentially KLH, and aluminum hydroxide asan adjuvant. Preferentially, 100 μg peptide/dose/patient (but also 1 μg,10 μg 100 μg and 1 mg will be applied in pre-clinical phase and 10 μg100 μg 200 μg in Phase I toxicity studies) will be applied once in 4weeks, 5 doses in total.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-10⁹, or more, virions perdose. Treatment can be monitored by assaying antibody, or activatedT-cell or B-cell responses to the therapeutic agent over time. If theresponse falls, a booster dose can be indicated.

Ultimately, the dosage regimen will be determined by the attendingphysician and by clinical factors. As is known in the medical arts,dosages for any one patient depend upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. A typical dose can be, forexample, in the range of 0.001 to 1000 mg; however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the regimen as a regularadministration of the pharmaceutical composition should be in the rangeof 1 mg to 10 mg units per day. If the regimen is a continuous infusion,it should also be in the range of 1 mg to 10 mg per kilogram of bodyweight per minute, respectively. Progress can be monitored by periodicassessment.

In addition, co-administration or sequential administration of otheragents can be desirable. In some embodiments, a therapeuticallyeffective dose or amount refers to that amount of the active ingredientsufficient to ameliorate the symptoms or condition. Therapeutic efficacyand toxicity of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose therapeutically effective in 50% of the population)and LD50 (the dose lethal to 50% of the population). The dose ratiobetween therapeutic and toxic effects is the therapeutic index, and itcan be expressed as the ratio, LD50/ED50. In some embodiments, thetherapeutic agent in the composition is present in an amount sufficientto restore normal behavior and/or cognitive properties in case ofAlzheimer's disease and related tauopathies.

The invention provides different measures that can be relied upon forevaluation of treatment effectiveness with any of the agents provided bythe invention. Examples include, but are not limited to, decreasedlevels of one or more pathological tau forms (e.g., within the brain),increased clearance of pathological tau from the brain and/or CSF;improved cognitive performance measures, such as cognitive functions(tested by, for example, Clinical Dementia Rating—CDR, Alzheimer'sdisease Assessment Scale—Cognitive Subscale—ADAS-Cog Mini Mental StateExamination—MMSE); improved motor function tests (e.g., Grip strengthtest, Timed Up & Go (TUG) test, TUG manual, Talking while Walking test,Unified Parkinson's disease Rating Scale—UPDRS); improved performance ofbasic activities of daily living (ADL) tests (e.g., hygiene, dressing,continence, eating, meal preparation, telephoning, going on an outing,finance, and correspondence; Disability Assessment in Dementia tests);and lessened severity/grading of AD impaired grip strength, locomotion,and apraxia (which have direct correlations with the animal models andassays described below, in the EXAMPLES), memory decline, aphasia,agnosia, disorientation in time and space, and depression.

For purposes of assessing treatment effectiveness, the levels anddistribution of tau (within the brain, and in body fluids) can beassayed by any of the methods described herein, and/or by any othermethods available to detect tau. For example, the levels of tau could bemeasured in vivo (Positron emission tomography) using novel imagingradiotracer 18F-THK523, which selectively binds tau and tau pathology invitro, ex vivo (tissue slices) and in vivo (transgenic mice)(Fodero-Tavoletti et al., 2011, Brain). Tau could be identified in thecerebrospinal fluid and in the blood as well using ELISA kitsrecognizing either total tau or phospho-tau.

Indeed, neurobehavioral impairment in transgenic rats has parallels withmotor impairment in Alzheimer's disease patients, which has implicationsfor clinical trials and treatment protocols with any of the therapeuticagents provided herein (including, but not limited to, agents for activevaccination. In humans, Alzheimer's disease is characterized clinicallyby progressive memory impairment and cognitive decline, behavioralchanges and psychological symptoms (disturbances in mood, emotion,appetite, wake sleep cycle, confusion, agitation and depression) andimpaired motor function (apraxia, myoclonus, gait impairment, decreasedmuscle strength, extrapyramidal features such as bradykinesia, rigidityand resting tremor) (Goldman et al., 1999; Boyle et al., 2009). Manystudies have reported that motor signs are commonly observed inAlzheimer's disease (AD) and become more prominent as the diseaseprogresses (Goldman et al., 1999; Wilson et al., 2003; Louis et al.,2004; Pettersson et al., 2005; Scarmeas et al., 2004; Scarmeas et al.,2005; Waite et al., 2005; Alfaro-Acha et al., 2006; Wang et al., 2006;Buchman et al., 2007a; Boyle et al., 2009). Notably, the motor signs canprecede the cognitive impairment and predict cognitive and functionaldecline, institutionalization and mortality in Alzheimer disease (Morriset al., 1989; Soininen et al., 1992; Kraemer et al., 1994; Chui et al.,1994; Scarmeas et al., 2004; Scarmeas et al., 2005). It has been shownthat decreased muscle strength precedes the development of cognitiveimpairment (Buchman et al., 2007b; Boyle et al., 2009).

The development of motor signs in AD has been associated with neuronaldegeneration and neuronal loss in the brainstem (Zarow et al. 2003;Burns et al. 2005; Grudzien et al. 2007; Simic et al., 2009; Wai et al.,2009; Braak and DelTredici, 2011). Moreover, several studies havesuggested that neurofibrillary degeneration originates in the brainstemand precedes cortical neurodegeneration (Hertz, 1989; Simic et al.,2009; Braak and DelTredici, 2011).

These findings show that motor impairment represents a key hallmark inAD pathogenesis. Moreover, functional impairment of some motor domainscan precede dementia and predict cognitive decline. Active immunotherapywith the peptides (including therapeutic epitopes) described herein canimprove motor impairment of transgenic rats expressing humanpathological tau. Thus, direct targeting of the brain stem pathology byactive immunotherapy can prevent, slow, or delay, motor as well ascognitive impairment in human AD patients. Thus, testing of motorfunctions can be included in the battery of tests that can be used forthe evaluation of the clinical efficacy of the agents (e.g., tauclearance agents, active and passive vaccines) described herein.

Also, one of ordinary skill in the art is aware of well-establishedcorrelations between the levels and distribution of pathological tau,(e.g., NFT in cortex/hippocampus) and disease progression. The densityof pathological tau (NFT pathology) has been correlated with cognitivedeficit and the severity of the Alzheimer's disease (Braak and Braak,1991; Bierer et al., 1995; Berg et al., 1998; Duyckaerts et al., 1998;Giannakopoulos et al., 1998, 2003). Pathological tau (e.g., NFTs,neuropil threads) in the entorhinal cortex and hippocampus are inverselyassociated with longitudinal changes in memory (Reitz et al., 2009).Similarly, in the brain stem, pathological tau (NFT) occurs in thedorsal raphe nucleus at a very early stage; the other raphe nuclei aresubsequently affected. These lesions explain the serotoninergic deficitfound in AD (Duykaerts et al., 2009). The extrapyramidal symptoms havebeen correlated with the substantia nigra tau pathology (Liu et al.,1997). Accordingly, a treatment agent that can affect one or more ofthese AD distribution patterns will likely have a beneficial effect inAD.

EXAMPLES Example 1 Preparation of Recombinant Human Tau Proteins

Human Full-Length Tau (2N4R, 2N3R) and Tau Deletion Mutants:

Tau recombinant proteins (FIGS. 1 and 6) were generated from clone τ40(Goedert, 1989), which was subcloned into the expression plasmid pET-17b(Novagen) and expressed in bacteria. Each tau deletion mutant wasverified by DNA-sequencing. All tau deletion mutants and tau peptidesare numbered according to the longest human tau isoform 2N4R, which is441 amino acids in length and thus is also called tau₄₄₁ (D'Souza,2005). Tau deletion mutants and peptides derived from the isoform 2N3Rare marked by “3R” to indicate that the second microtubule bindingrepeat (amino acids 275-305 of 2N4R) is missing. Production of tauproteins involved the following steps: a) expression of tau in bacteria;b) tau purification by ion exchange chromatography; c) tau purificationby gel-filtration; d) concentration and storage of isolated tau; and e)immunoaffinity purification (this is an exception adopted only fortauΔ(1-150;392-441)/4R, which was used in the microglia uptakeexperiments, see Example 10, FIG. 17).

a) Bacterial Expression of human full-length tau (either 2N4R or 2N3R)and recombinant tau deletion mutants: human tau (above) expressionplasmids were transformed into Escherichia coli (E. coli), productionstrain BL21(DE3). Bacterial cells containing the appropriate expressionplasmid were cultivated and induced as described in “Molecular Cloning:A Laboratory Manual” by Sambrook and Russell (2001). A single colony ofBL21(DE3) bacteria, transformed with pET-17b plasmid driving expressionof a tau protein or its fragment, were grown at 37° C. in 500 ml ofLuria broth medium with 100 μg/ml ampicillin at 300 rpm and induced bythe addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG) to a finalconcentration of 0.4 mM. After further incubation at 37° C. for 3 hours,bacteria were collected by centrifugation at 3,000×g for 15 min at 4° C.

b) Cation-exchange chromatography purifications of the basic and neutraltau proteins (full-length tau isoforms, tauΔ358-441, tauΔ306-400,tauΔ421-441, tauΔ300-312, tauΔ134-168, tauΔ1-220, tauΔ1-126, tauΔ(1-150;392-441)/4R, tauΔ(1-150; 392-441)/3R and tauΔ(1-296;392-441)/4R) weredone essentially as previously described (Krajciova et al., 2008). Afterexpression, the bacterial pellets were resuspended in 10 ml of lysisbuffer (50 mM 1,4-piperazinediethanesulfonic acid (PIPES) pH 6.9, 50 mMsodium chloride (NaCl), 1 mM ethylenediaminetetraacetic acid (EDTA), 5mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 5%(v/v) glycerol), quickly frozen in liquid nitrogen, and stored at −80°C. until used for purification of tau proteins. For tau proteinpurification, the frozen bacterial suspensions were quickly thawed andplaced on ice. Bacterial cell walls were broken by sonication on ice byusing Sonopuls HD 2200, tip TT-13 (Bandelin, Germany) set to 50% dutycycle, 50 W power output, 6 times for 30 s with 30 s pauses. The lysateswere clarified by centrifugation (21,000×g for 15 min at 4° C.) and thesupernates were filtered through a 0.45 μm membrane filter. Large-scalepurification of the recombinant tau proteins was done at 6° C. using anÄKTA-FPLC workstation (Amersham Biosciences, Sweden). The filteredlysates were loaded at a 3 ml/min flow rate onto a 5-ml HiTr ap SP HPcolumn (GE Healthcare, Uppsala, Sweden) equilibrated with the lysisbuffer, and washed extensively with 60 ml of the lysis buffer until thebaseline at 280 nm became stable. Bound tau proteins were eluted by agradient (0-30% within 15 ml) of Buffer B (lysis buffer supplementedwith 1 M NaCl). Individual 1 ml fractions were collected and analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Toremove nucleic acids, which copurify with positively charged tauproteins, the fractions containing tau protein were pooled and purifiedby a second cation-exchange chromatography step, using a 5-ml HiTrap SPHP column (GE Healthcare, Uppsala, Sweden) with a less steep gradient ofBuffer B (0-30% in 45 ml).

c) Anion-exchange chromatography purification of the acidic tau proteins(tauΔ222-427, tauΔ228-441, tauΔ257-400, tauΔ137-441, tauΔ283-441) wasdone as previously described (Csokova et. al 2004). After expression,bacterial pellets were resuspended in 10 ml of histidine lysis buffer(20 mM histidine, pH 6.0, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, 0.1 mM PMSF,and 5% (v/v) glycerol). Bacterial cell walls were broken by sonicationon ice by using Sonopuls HD 2200, tip TT-13 (Bandelin, Germany) set to50% duty cycle, 50 W power output, 6 times for 30 s with 30 s pauses.The lysates were clarified by centrifugation (21,000×g for 15 min at 4°C.). Bacterial lysates were precipitated by 1% streptomycin sulfate(Medexport, Russia), incubated on ice for 5 min, clarified bycentrifugation (21,000×g for 15 min at 4° C.), and filtered through a0.45 μm membrane filter. The filtered streptomycin precipitated lysateswere loaded at 3 ml/min flow rate onto a 5 ml HiTrap QSepharose HPcolumn (Amersham Biosciences, Sweden) and washed extensively with 30-50ml histidine lysis buffer until the A280 baseline became stable. Tauproteins were eluted with a two-step salt gradient (0.05-0.5M NaCl in 40ml followed by 0.5-1 M NaCl in 20 ml) in histidine lysis buffer.

d) In the final gel-filtration step of purification (the same for alltau proteins), pooled tau protein fractions obtained by ion exchangechromatography, were injected onto a gel-filtration column (HiLoad 26/60Superdex 200 prep grade column, GE Healthcare) at 3 ml/min in eitherPIPES or Histidine lysis buffer for basic/neutral or acidic tauproteins, respectively, supplemented with 100 mM NaCl. Eluted tauproteins were pooled.

e) For tau protein concentration after gel-filtration purification,pooled fractions were diluted with 1.5 volumes of 2.5% glycerol, andloaded again on a HiTrap SP HP column (basic and neutral tau proteins)or on a HiTrap Q HP column (acidic tau proteins). The concentratedrecombinant tau protein was then eluted from the column with a 1 M NaClstep gradient. Finally, the buffer was exchanged to phosphate-bufferedsaline (PBS, 8.09 mM disodium phosphate (Na₂HPO₄), 1.47 mM potassiumdihydrogen phosphate (KH₂PO₄), 136.89 mM NaCl, 2.7 mM potassium chloride(KCl)) saturated with argon, using a 5 ml HiTrap Desalting column (GEHealthcare). Protein quantitation of purified samples was done usingbicinchoninic acid (BCA) quantitation kits (Pierce, USA), with bovineserum albumin (BSA) as a standard. Tau proteins were aliquoted intoworking aliquots, snap-frozen in liquid nitrogen, and stored at −70° C.

f) In order to remove possible bacterial contaminants from therecombinant tauΔ(1-150;392-441)/4R used for the measurements of tauuptake by microglia (Example 10, FIG. 17), the recombinant tau proteinwas purified by a modified method, as follows. After the firstcation-exchange chromatography step, the fractions containing tau werepooled and 1/20 volume of ice-cold 5% polyethylenimine was added whilestirring. The stirring continued for another 30 min, on ice. The samplewas centrifuged at 20,000×g for 15 min at 4° C. The supernate wascollected and injected onto a HiLoad 26/60 Superdex 200 prep gradecolumn (GE Healthcare) at 3 ml/min in the PIPES lysis buffersupplemented with 100 mM NaCl but lacking DTT or any other reducingagent. After gel filtration, fractions with tau protein were pooled andloaded onto an immunoaffinity column (at a flow rate of 0.5 ml/min)containing DC25 antibody (epitope 347-353 of 2N4R tau, AxonNeuroscience, Vienna, Austria) immobilized on CNBr-activated Sepharose.The column was pre-equilibrated in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl,0.1% Tween 20 (TBS-Tween). After binding, the column was washed with 5column volumes of TBS-Tween and the bound tau proteins were eluted with0.1 M glycine, pH 2.7. The collected fractions were neutralized byadding 1/30 volume of 1 M Tris-HCl pH 8.8 and pooled. Lastly, the bufferwas exchanged to PBS (saturated with argon), using an HiTrap Desaltingcolumn, 5 ml (GE Healthcare). Protein quantitation of purified sampleswas done using a bicinchoninic acid (BCA) quantitation kit (Pierce,USA), with BSA as a standard. The protein was aliquoted into workingaliquots, snap-frozen in liquid nitrogen, and stored at −70° C.

The purified DC25 antibody (Axon Neuroscience, Vienna, Austria) used forthe DC25 affinity column (supra) was prepared as follows. Serum freeDC25 hybridoma culture supernate was adjusted to pH 7.5 by adding 0.2volume of PBS, precleared by centrifugation at 20,000×g for 10 minutesat 4° C., and the supernate filtered through a 0.2 μm filter. Thepre-cleared DC25 hybridoma culture supernate was loaded onto aPBS-equilibrated HiTrap Protein G HP column (5 ml, GE Healthcare) at 1ml/min. After loading was complete, the column was washed with 4 columnvolumes of PBS, and the bound antibody was eluted with 100 mM glycine pH2.7. Eluted fractions were neutralized with 1 M Tris-HCl pH 9, pooled,and buffer exchanged into PBS using a HiTrap Desalting column (5 ml, GEHealthcare). The purified DC25 antibody was stored in small aliquots at−70° C.

Example 2 Preparation of Hybridoma Cell Lines Producing MonoclonalAntibodies Against Human TauΔ(1-150;392-441)/4R, Screening of MonoclonalAntibodies by ELISA, and Initial Characterization of Monoclonal AntibodyDC8E8

Six-week-old Balb/c mice were primed subcutaneously with 50 μg ofrecombinant tauΔ(1-150;392-441)/4R (prepared as described in Example 1)in complete Freund's adjuvant (SIGMA), and boosted five times atfive-week intervals with 50 μg of the same antigen in incompleteFreund's adjuvant. Three days before the fusion, mice were injectedintravenously with 50 μg of the same antigen in PBS. Spleen cells fromimmunized mice were fused with NS/0 myeloma cells according to themethod of Kontsekova et al. (1988). Splenocytes (10⁸) were mixed with2×10⁷ NS/0 myeloma cells (ratio 5:1) and fused for 1 minute in 1 ml of50% polyethylene glycol (PEG) 1550 (Serva) in serum free Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% dimethylsulphoxide. The fused cells were resuspended in DMEM containing 20%horse serum, L-glutamine (2 mM), hypoxanthine (0.1 mM), aminopterin(0.04 mM), thymidine (0.016 mM), and gentamycin (40 U/ml), at a densityof 2.5×10⁵ spleen cells per well on 96-well plates. The cells wereincubated for 10 days at 37° C. and growing hybridomas were screened forthe production of anti-tauΔ(1-150;392-441)/4R-specific monoclonalantibodies by an enzyme-linked immunosorbent assay (ELISA).

An ELISA was used to detect monoclonal antibodies in hybridoma culturesupernates directed against tauΔ(1-150;392-441)/4R (a misdisordered formof tau). Microtiter plates were coated overnight withtauΔ(1-150;392-441)/4R (5 μg/ml, 50 μl/well) at 37° C. in PBS. Afterblocking with 1% nonfat dried milk to reduce nonspecific binding, theplates were washed with PBS-0.05% Tween 20 and incubated with 50 μl/wellof hybridoma culture supernate for 1 hr at 37° C. Bound monoclonalantibodies were detected with sheep anti-mouse immunoglobulin (Ig)conjugated with horse radish peroxidase (HRP, DAKO). The reaction wasdeveloped with orthophenylenediamine solution as a peroxidase substrateand stopped with 50 μl of 2 M H₂SO₄. Absorbance at 492 nm was measuredusing a Multiscan MCC/340 ELISA reader (Labsystems). Readouts with anabsorbance value of at least twice the value of the negative controls(PBS) were considered positive. Positive hybridoma cultures were furthertested by immunohistochemistry (accordingly to method of Zilka et al.,2003) and subcloned in soft agar according to the procedure described inKontsekova et al. (1991).

The monoclonal antibody DC8E8 (produced by the mouse hybridoma cell linedeposited with the American Type Culture Collection on Jul. 13, 2011,with the ATCC Patent Deposit Designation PTA-11994) was identified amongthe positive hybridoma cultures so produced and selected. DC8E8 wasfurther characterized as described below. The antibody isotype wasdetermined to be murine IgG1 by ELISA using a mouse Ig isotyping kit(ISO-2, SIGMA).

Example 3 Sequencing of Variable Regions of DC8E8 and its Humanizationby CDR-Grafting

a) Determination of the nucleotide and amino acid sequences of the lightand heavy chain variable regions of DC8E8 (FIG. 3). The nucleotidesequence of DC8E8's variable regions (FIGS. 3A and 3D) was determined byDNA sequencing of cDNA synthesized using total RNA extracted from themouse hybridoma cell line PTA-11994 (ATCC), which expresses the DC8E8monoclonal antibody. Total RNA was extracted using TRIZOL® Reagent(Invitrogen, USA). Synthesis of the first strand cDNA was carried outusing the “High capacity cDNA reverse transcription” kit according tothe manufacturer's protocol (Applied Biosystems, USA). The compositionof the reagents for the 2× reverse transcription master-mix was asfollows (quantities per 20 μL reaction): 2 μL of 10×RT buffer; 0.8 μL of25×dNTP Mix (100 mM); 2 μl of 10×RT Random Primers (50 μM); 1 μL ofMultiScribe™ Reverse Transcriptase (50 U/μL); 4.2 μL of nuclease-freeH₂O. For reverse transcription, 10 μL of the 2× reverse transcriptionmaster-mix was mixed with RNA sample (2 μg/10 μL) and cDNA wassynthesized under the following conditions: 10 min at 25° C., 120 min at37° C., 5 min at 85° C., and final cooling to 4° C. Amplification of thegenes encoding the variable regions of the light and heavy chains wasdone by polymerase chain reaction (PCR) using Phusion® High-Fidelity DNAPolymerase (Finnzymes, Finland). The forward primers (8E8L-sense5′-ACATTGTGATGTCACAGTCTCCATCCTCC-3′ (SEQ ID NO: 132) and 8E8H-sense5′-CTCCTCCAATTGCAGCAGTCTGG-3′(SEQ ID NO: 133)) were designed accordingto the protein sequence of the N-terminal ends of DC8E8 light(DIVMSQSPSS) (SEQ ID NO: 134) and heavy (QVQLQQSGPE) (SEQ ID NO: 135)chains. The N-terminal protein sequences were determined using Edmandegradation (light chain) and MALDI in-source decay (heavy chain). Usingthis information, the most similar proteins to the light and heavychains were identified in the Genebank along with their correspondingnucleotide sequences. The most probable nucleotide sequences of themouse V-genes (light and heavy) were then identified in the IMGT/LIGM-DBdatabase (www.imgt.org). These genes were used for the design of theforward primers (corrections were made using the N-terminal proteinsequences of DC8E8). The reverse primers for the light and heavy chains(Kappa-antisense 5′-GGAATTCGTTGAAGCTCTTGACAATGGGTG-3′ (SEQ ID NO: 136)and G1-antisense 5′-GGAATTCACATATGCAAGGCTTACAACCAC-3 (SEQ ID NO: 137))were derived from kappa and IgG1 chains constant regions, respectively.

The PCR products were sequenced and the resulting DNA sequences ofvariable regions of light and heavy chains of DC8E8 are shown in FIGS.3A and 3D, respectively. The alignment of DC8E8 to the closest mousegermline light chain IGKV8-21*01 and heavy chain IGHV1-81*01 are shownin FIGS. 3C and 3F, respectively. Complementarity determining regions(CDRs) are underlined in the DC8E8 light and heavy chains proteinsequences (FIGS. 3B and 3E, respectively). CDRs and framework regions(FR) were identified according to the ImMunoGeneTics (IMGT) numberingsystem (see, e.g., Lefranc M. P. The IMGT unique numbering forimmunoglobulins, T-cell receptors, and Ig-like domains. The Immunologist7, 132-136, 1999 (1999)).

b) Humanization of DC8E8. To identify a suitable candidate humanimmunoglobulin for production of a humanized DC8E8 through grafting ofthe mouse DC8E8 complementarity determining regions (CDRs), the humangermline gene with the highest sequence identity to DC8E8 was determinedusing ClustalX2 pairwise alignment of the DC8E8 nucleotide sequenceagainst a selected set of human immunoglobulin genes extracted fromIMGT/LIGM-DB flat file release 201112-6 (www.imgt.org). IgKv4-1*01 wasidentified as the closest human germline gene for the DC8E8 light chain(FIG. 4), and IgHV1-6910 was identified as the closest germline gene forthe DC8E8 heavy chain (FIG. 5). The following approach (Method 1 andMethod 2) was designed and can be used to prepare one or more humanizedversions of the DC8E8 antibody. After expression in an appropriateantibody expression system (e.g., mammalian expression vectors used forantibody expression in vitro (e.g., HEK293 cells) or in vivo (transgenicanimals)), the resulting humanized, recombinant antibodies, can betested for activity (e.g., biochemical and therapeutic activity)according to any of the methods used for characterization of DC8E8'sactivity.

Method 1: CDR grafting and mutations in the framework region (FR), ifnecessary (CDRs are in bold underlined, FR mutations are in bold):

Heavy chain variable region (SEQ ID NOS 138-140,respectively, in order of appearance): DC8E8_heavyQVQLQQSGPELVKPGTSVKMPCKAS GYIFTDYV IS WVKQRTGQGLEWIGE IFPRSGST YYhuman_germ_heavyQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPILGIANY**** ***.*: ***:***:.*****  *:.*.****:* .******:* *:*  * : *SEQ ID No. 140 QVQLVQSGPEVKKPGSSVKVPCKAS GYIFTDYVI S WVRQATGQGLEWMGEIFPRSGST NY DC8E8_heavy NEKFKGKATLTADKSSNTAYMQLSSVTSEDSAVYFCARDYYGTSFAMDY WGQGTSVTVSS human_germ_heavyAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENHCYYYGMDVWGQGTTVTVSS :**:*:.*:*****:.****:***: ***:***:***: :   :.** *****:*****SEQ ID No. 140 AQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC ARDYYGTSFAMDYWGQGTTVTVSS Light chain variable region (SEQ ID NOS 141-143,respectively, in order of appearance): DC8E8_lightDIVMSQSPSSLAVSAGEKVTMSCKSS QSLLNSRT RKNY LAWYQQKPGQSPKLLIY WAS TRhuman_germ_lightDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTR****:***.***** **:.*:.******:* * ..*************.***********SEQ ID No. 143 DIVMTQSPDSLAVSLGERATINCKSS QSLLNSRT RKNYLAWYQQKPGQSPKLLIY WAS TR DC8E8_light ESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSFYLRT FGGGTKLDIK human_germ_lightESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTLTFGGGTKVEIK********:**************:****:*****:* :   *******::** SEQ ID No. 143ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC KQSFYLRT FGGGTKVEIK

Method 2: The mouse (FIG. 3) and human (FIGS. 4 and 5) germlineimmunoglobulins with the highest sequence identity with DC8E8 were foundand aligned to the DC8E8 protein sequence. The CDR regions wereidentified following the IMGT numbering system. The most probableantigen-contacting residues within the DC8E8 combining site wereidentified on the basis of the work of MacCallum et al, J. Mol. Biol.1996.

Various amino acid candidates for mutation in the humanized version ofDC8E8 were identified on the basis of the following combined criteria:

-   -   i. their presence in the CDR and probability of contact with        antigen    -   ii. their presence in the Vernier zone    -   iii. whether or not they were mutated in the mouse germline

Two levels of mutation candidates were identified according to the abovecriteria:

X-Type Residues (in Bold):

-   -   Residues different between DC8E8 and the closest mouse germline,        non-similar amino acids    -   Residues in the CDR and contacting antigen. CDRs are in        lowercase bold italic in the DC8E8 sequence below.

Y-Type Residues (in Bold Underlined):

-   -   Residues identical between DC8E8 and the closest mouse germline,        but different in the closest human germline and located in the        Vernier zone (non-similar amino acid)    -   Residues different between DC8E8 and the closest mouse germline        (similar/conserved amino acid)

Two humanized sequences for each chain were identified with mutationspredicted to affect DC8E8's activity:

-   -   SEQ ID Nos.147, 152: Only X type of residues will be mutated    -   SEQ ID Nos.148, 153: Both X and Y type of residues will be        mutated

Heavy chain variable region (SEQ ID NOS 138, 145,139, respectively, in order of appearance): DC8E8_heavyQVQLQQSGPELVKPGTSVKMPCKAS

WVKQRTGQGLEWIGE

YY mouse_germ_heavyQVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYhuman_germ_heavyQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPILGIANY**** ***.*: :**:***:.*****  *:.* ****:* .******:* * *  * : *SEQ ID No. 147QVQLVQSGPEVVKPGSSVKMPCKASGYIFSDYAISWVRQRTGQGLEWMGEIFPRSGSTNYSEQ ID No. 148QVQLVQSGPEVVKPGSSVKMPCKASGYIFSDYAISWVRQRTGQGLEWMGEIFPRSGST Y YDC8E8_heavy NEKFKGKATLTADKSSNTAYMQLSSVTSEDSAVYC

WGQGTSVTVSSF mouse_germ_heavyNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARDYYGTYYAMDYWGQGTSVTVSShuman_germ_heavyAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENHCYYYGMDVWGQGTTVTVSS :**:*:.*:*****:.****:* *: ***:***:***: :   :.** *****:*****SEQ ID No. 147AQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDYYGTSYGMDVWGQGTTVTVSSSEQ ID No. 148 N QKFQGRVTITADKST N TAYM Q LSSL TSEDTAVYYCARDYYGTSYGMDVWGQGTTVTVSSLight chain variable region (SEQ ID NOS 141, 150,142, respectively, in order of appearance): DC8E8_lightDIVMSQSPSSLAVSAGEKVTMSCKSS

LAWYQQKPGQSPKLLIY

TR mouse_germ_lightDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRhuman_germ_lightDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTR****:***.***** **:.*:.******:* * ..*************.***********SEQ ID No. 152DIVMTQSPDSLAVSLGERATINCKSSQSVLNSRNNKNYLAWYQQKPGQPPKLLIYWASTRSEQ ID No. 153 DIVMTQSPDSLAVSLGERATI S CKSSQSVLNSRNNKNYLAWYQQKPGQ SPKLLIYWASTR DC8E8_light ESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC

FGGGTKLDIK mouse_germ_lightESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLRTFGGGTKLEIK human_germ_lightESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTLTFGGGTKVEIK********:**************:****:*****:* :   *******::** SEQ ID No. 152ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSFYLRTFGGGTKVEIK SEQ ID No. 153ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSFYLRTFGGGTKVEIK

Example 4 Mapping of the Dc8E8 Epitope Using Recombinant Tau DeletionMutants and Tau-Derived Peptides

Deletion mutants of human tau protein 2N4R, as well as tau derivedpeptides (Antagene, Inc. (Sunnyvale, Calif.) and EZBiolab, (USA)) wereused for epitope mapping of DC8E8 using ELISA (FIGS. 6, 7, and 8).Recombinant human tau isoforms (2N4R; 2N3R) and tau deletion mutants(FIGS. 6A, 6B) were prepared as described in Example 1. Peptides (FIG.7A, 7B) were synthesized by EZBiolabs (USA) with purity higher than 85%.

Microtiter plates were coated overnight at 37° C. with eitherrecombinant tau proteins or with tau peptides (5 μg/ml in PBS, 50μl/well). After blocking with 1% nonfat dried milk to reduce nonspecificbinding, the plates were washed with PBS-0.05% Tween 20 and incubatedwith 50 μl/well of DC8E8 hybridoma culture supernate, for 1 hr at 37° C.Bound monoclonal antibody was detected with sheep anti-mouse IgHRP-conjugated (DAKO). The reaction was developed withorthophenylenediamine solution as a peroxidase substrate and stoppedwith 50 μl of 2 M H₂SO₄. Absorbance was measured at 492 nm using aMultiscan MCC/340 ELISA reader (Labsystems). Readouts with an absorbancevalue of at least twice the value of the negative controls (PBS) wereconsidered positive.

DC8E8 recognized the following human tau proteins: Δ58-441, Δ421-441,Δ134-168, Δ1-220, Δ1-126, Δ(1-296;392-441)/4R and Δ(1-150;392-441)/4R,but failed to recognize the tau proteins with deletions Δ222-427,Δ306-400, Δ228-441, Δ300-312, Δ257-400, Δ137-441, and Δ283-441 (FIG. 6B,6C). DC8E8 recognized the physiological tau isoforms 2N4R and 2N3R to alesser extent than it recognized the pathological/misdisorderedtauΔ(1-296;392-441)/4R, tauΔ(1-150;392-441)/4R and the tau deletionmutants (A358-441, Δ421-441, Δ134-168, Δ1-220, Δ1-126) of tau 2N4R (FIG.6C). More detailed epitope mapping, using tau peptides, revealed thatDC8E8 did not recognize tau peptides 240-270, 270-300, and 301-330 (FIG.7A, 7B, 7C). Together, these findings suggest that DC8E8 has fourbinding sites or epitopes on human tau, each of which is located in themicrotubule-binding repeat domain region of the tau protein, and each ofwhich epitopes is separately located within one of the following tausequences: 267-KHQPGGG-273 (SEQ ID NO: 98) (1st repeat domain of tauprotein), 298-KHVPGGG-304 (SEQ ID NO: 99) (2nd repeat domain of tauprotein), 329-HHKPGGG-335 (SEQ ID NO: 100) (3rd repeat domain of tauprotein), and 361-THVPGGG-367 (SEQ ID NO: 101) (4th repeat domain of tauprotein) (FIG. 7D). Moreover, because DC8E8 binds to the truncated formsof tau better than to the full-length 3-repeat and 4-repeat tau, theseresults also suggest that DC8E8 binds better to disease forms of tauthan to physiological tau (tau39 (2N3R) and tau40 (2N4R)). Also, becausetau is thought to change conformation from physiological tau(intrinsically disordered) to disease tau (misdisordered and misordered,Kovacech et al., 2010), these results suggest that one or more of thebinding sites for DC8E8 (the DC8E8 epitopes) has a differentconformation in physiological tau than it does in disease tau, and thatDC8E8 is capable of detecting that conformational change.

Because these tau repeat domains are conserved across species (FIG. 8A),DC8E8 is likely to react against tau proteins from such diverse speciesas rat, mouse, cow, chipanzee, frog, and others. An alignment of tauproteins of various animal species was done using software ClustalW2(available, for example, at www.ebi.ac.uk/Tools/msa/clustalw2/). Humantau is represented by the longest tau isoform expressed in human brainneurons (2N4R, 441 amino acids). Tau proteins of other species wereselected from public databases. The sequences within which each of thefour epitopes recognized by DC8E8 antibody is located are boxed.

Additional point mutations and deletions were done on certaintau-derived peptides (8-mers, 9-mers, and 10-mers) to further define theDC8E8 epitopes, as assessed by each peptide's ability to compete withtauΔ(1-150;392-441/4R) for binding to DC8E8. Peptides were synthesizedby EZBiolabs (USA) with purity higher than 85%. The competition ELISAwas carried out according to the following standard protocol. ELISAplates (IWAKI high bind plate, #3801-096, Bertoni GmbH, Austria) werecoated overnight at 4° C. with 100 μl/well of 5 μg/ml of recombinantpurified tauΔ(1-150;392-441/4R) in PBS. The IWAKI high bind plates werewashed 4 times with PBS/Tween 20 (0.05% v/v), and blocked with PBS/Tween20 for 2 h at 25° C. Each of the peptides was separately dissolved inPBS at a final concentration of 5 mM. Serial dilutions (2-fold) of thepeptides in PBS/Tween 20 were prepared in polypropylene plates withconical well bottom (Greiner, #651201) (concentration range 80 μM, 40μM, 20 μM, 10 μM, 5 μM, and 2.5 μM). 100 μl of each dilution were addedper well. Purified DC8E8 monoclonal antibody (purification was done asdescribed below in Example 5) was diluted to a concentration of 2 μg/mlin PBS/Tween 20 and 100 μl of this diluted antibody was mixed with eachserial dilution of peptides resulting in 200 μl mixtures with 100 ng ofantibody/100 μl containing each respective test peptide at aconcentration of 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM. Theantibody/peptide mixtures were incubated for 1 hr at 25° C. on arotating platform set to 250 rpm. One hundred microliters (100 μl) ofantibody/peptide mixtures were transferred from the polypropylene platesinto tauΔ(1-150;392-441/4R)-coated and PBS/Tween 20-blocked IWAKI highbind plates, and incubated for 1 hr at 25° C. on a rotating platform setto 250 rpm. The plates were washed 4× times with PBS/Tween 20. Thesamples (in the plates) were incubated for 1 hr at 25° C. on a rotatingplatform (set to 250 rμm) with 100 μl of Polyclonal Goat Anti-MouseImmunoglobulins/HRP (Dako, #P0447) diluted 1:4,000 in PBS/Tween 20. Theplates were washed 4× times with PBS/Tween. The samples/plates were thenincubated with 100 μl of a 1.5 mg/2 ml solution of o-PDA(o-phenylenediamine, SIGMA, P1526) in 0.1 M Na-Acetate pH 6.0 (Roth,#6779) supplemented with 1.5 μl/2 ml of 30% H₂O₂ (SIGMA, H-0904) for 10minutes at 25° C., in the dark. The reaction was stopped by adding 100μl of 2 M H₂SO₄ (Merck, 1.00731.1000). The extent of reaction wasfollowed by reading the absorbance of the samples/plates at 490 nm (e.g.using the Victor Multilabel Counter (Wallac).

FIG. 8B shows the results of the competition ELISA performed with thefollowing six peptides: NIKAVPGGGS (SEQ ID NO: 200), NIKHVPGGGS (SEQ IDNO: 201), IKHVPGGGS (SEQ ID NO: 202), KHVPGGGSV (SEQ ID NO: 203),HVPGGGSVQ (SEQ ID NO: 204), and VPGGGSVQ (SEQ ID NO: 205). The peptidesKHVPGGGSV (SEQ ID NO: 203) and HVPGGGSVQ (SEQ ID NO: 204), encompassingtau therapeutic epitope #2, competed with at least one of the originaltherapeutic epitopes present on tauΔ(1-150;392-441/4R). Removal of theunderlined histidine from the epitope of SEQ ID NO: 204 lead to a lossof competing activity (see peptide VPGGGSVQ, SEQ ID NO: 205). A pointmutation changing a histidine to alanine (at corresponding tau position299, in “epitope #2”) lead to a loss of competing activity (peptideNIKAVPGGGS, SEQ ID NO: 200). Peptides containing 2 or 3 amino acidsbefore “histidine 299” (towards the N terminus) also competed with theoriginal epitope (peptides IKHVPGGGS (SEQ ID NO: 202) and NIKHVPGGGS(SEQ ID NO: 201), respectively). These results suggest that the minimalepitope of DC8E8 falling within the second tau repeat (epitope #2) iswithin a 6-mer sequence, namely HVPGGG (SEQ ID NO: 154).

The aforementioned mapping experiments suggested the presence of theamino acid sequence PGGG within one ore more of epitopes of the DC8E8antibody. Furthermore, this amino acid sequence is present in all fourepitopes on tau protein bound by DC8E8 (see SEQ ID NOs: 98, 99, 100,101). In order to determine the residues in the N-terminal region of theDC8E8 epitopes, alanine scanning experiments were done on tau peptide295-DNIKHVPGGGS-305, which comprises the DC8E8 epitope (within298-KHVPGGG-304, SEQ ID NO: 99) that falls within the 2^(nd) repeatdomain of tau.

The binding capacity of the mutant peptides to DC8E8 was assessed byeach peptide's ability to compete with tauΔ(1-150;392-441/4R) forbinding to DC8E8. Seven peptides were synthesized by EZBiolabs (USA)with purity higher than 85%: ANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS(SEQ ID NO: 146), DNAKHVPGGGS (SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO:151), DNIKAVPGGGS (SEQ ID NO: 159), DNIKHAPGGGS (SEQ ID NO: 161), andthe peptide with the original sequence DNIKHVPGGGS (SEQ ID NO: 171). Thecompetition ELISA was carried out according to the following standardprotocol. ELISA plates (IWAKI high bind plate, #3801-096, Bertoni GmbH,Austria) were coated overnight at 4° C. with 100 μl/well of 5 μg/ml ofrecombinant purified tauΔ(1-150;392-441/4R) in PBS. The IWAKI high bindplates were washed 4 times with PBS/Tween 20 (0.05% v/v), and blockedwith PBS/Tween 20 for 2 h at 25° C. Each of the peptides was separatelydissolved in PBS at a final concentration of 5 mM. Serial dilutions(2-fold) of the peptides in PBS/Tween 20 were prepared in polypropyleneplates with conical well bottom (Greiner, #651201) (concentration range320 μM, 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, and 2.5 μM). 100 μl ofeach dilution were added per well. Purified DC8E8 monoclonal antibody(purification was done as described below in Example 5) was diluted to aconcentration of 2 μg/ml in PBS/Tween 20 and 100 μl of this dilutedantibody was mixed with each serial dilution of peptides resulting in200 μl mixtures with 100 ng of antibody/100 μl containing eachrespective test peptide at a concentration of 160 μM, 80 μM, 40 μM, 20μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM. The antibody/peptide mixtures wereincubated for 1 hr at 25° C. on a rotating platform set to 250 rpm. Onehundred microliters (100 μl) of antibody/peptide mixtures weretransferred from the polypropylene plates intotauΔ(1-150;392-441/4R)-coated and PBS/Tween 20-blocked IWAKI high bindplates, and incubated for 1 hr at 25° C. on a rotating platform set to250 rpm. The plates were washed 4× times with PBS/Tween 20. The samples(in the plates) were incubated for 1 hr at 25° C. on a rotating platform(set to 250 rμm) with 100 μl of Polyclonal Goat Anti-MouseImmunoglobulins/HRP (Dako, #P0447) diluted 1:4,000 in PBS/Tween 20. Theplates were washed 4× times with PBS/Tween. The samples/plates were thenincubated with 100 μl of a 1.5 mg/2 ml solution of o-PDA(o-phenylenediamine, SIGMA, P1526) in 0.1 M Na-Acetate pH 6.0 (Roth,#6779) supplemented with 1.5 μl/2 ml of 30% H₂O₂ (SIGMA, H-0904) for 10minutes at 25° C., in the dark. The reaction was stopped by adding 100μl of 2 M H₂SO₄ (Merck, 1.00731.1000). The extent of reaction wasfollowed by reading the absorbance of the samples/plates at 490 nm (e.g.using the Victor Multilabel Counter (Wallac).

FIG. 8C shows the results of the competition ELISA performed with thefollowing seven peptides: ANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS (SEQID NO: 146), DNAKHVPGGGS (SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO: 151),DNIKAVPGGGS (SEQ ID NO: 159), DNIKHAPGGGS (SEQ ID NO: 161), andDNIKHVPGGGS (SEQ ID NO: 171). A point mutation changing a histidine toalanine (at corresponding tau position 299, in “epitope #2”) lead to acomplete loss of competing activity with tauΔ(1-150;392-441/4R) forbinding to DC8E8 (peptide DNIKAVPGGGS, SEQ ID NO: 159). Mutations thatchanged amino acids D, N, I, K and V to alanine did not abolish thecompeting activity of the respective mutant peptides (peptidesANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS (SEQ ID NO: 146), DNAKHVPGGGS(SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO: 151), DNIKHAPGGGS (SEQ ID NO:161). These results suggest that the minimal epitope of DC8E8 fallingwithin the second tau repeat (epitope #2) is within a 6-mer sequence,namely HVPGGG (SEQ ID NO: 154), and that DC8E8 binds to HXPGGG (SEQ IDNO:164).

Example 5 DC8E8 Recognizes Misdisordered TauΔ(1-150;151-391)/4R, asAssessed by Surface Plasmon Resonance

Surface plasmon resonance (SPR) can be used for the detection of proteinbinding and to determine the thermodynamic parameters of proteincomplexes (e.g., antibody-antigen complexes) by direct monitoring of thebinding event in real time. This technology is routinely used tocharacterize both diagnostic and therapeutic antibodies (See, e.g.,Karlsson and Larsson, Affinity Measurement Using Surface PlasmonResonance, in Methods in Molecular Biology, Vol. 248: AntibodyEngineering: Methods and Protocols. Edited by: B. K. C. Lo © HumanaPress Inc., Totowa, N.J., (2008)).

For SPR experiments, the DC8E8 monoclonal antibody (mAb) was purifiedfrom serum-free hybridoma supernate on a Protein G affinity column, asfollows. The hybridoma supernate was adjusted to pH 7.5, the solutionwas pre-cleared by centrifugation, filtered through a 0.45 μm membranefilter, and loaded onto a 5 ml Protein G Sepharose column. DC8E8 mAb waseluted from the column with 0.1 M Glycine-HCl, pH 2.7. Eluted fractionswere immediately neutralized with 1M Tris-HCl pH 9.0. Pooled fractionswere dialyzed against PBS, concentrated by ultrafiltration, and storedat −70° C. The concentration of the antibody was determined by measuringabsorbance at 280 nm, using the formula c(mg/ml)=A_(280nm)/1.43.

A BIACORE3000 instrument with a CM5 sensor chip (Biacore AB, Uppsala)was used for the SPR assays. Amine-coupling reagents (EDC, NHS,ethanolamine pH 8.5), P20 detergent, and 10 mM sodium acetate pH 5.0were obtained from Biacore AB. These experiments were done at 25° C. inPBS pH 7.4 with 0.005% of P20 (PBS-P) as the running buffer. Typically,5,000 RU (response units) of polyclonal anti-mouse antibody (No. Z 0420;DakoCytomation, Glostrup, Denmark) was coupled at pH 5.0 via primaryamines simultaneously in two flow cells, one of which was used as areference measurement.

In each analysis cycle, purified DC8E8 was captured in the analyticalflow cell to reach an immobilization level of 230-250 RU. For K_(A)determinations, as well as for the determination of kinetic rateconstants (k_(ON) and k_(OFF)), two-fold serial dilutions of either tauproteins (against which DC8E8 affinity was tested), or PBS-P as acontrol, were injected at a flow rate 50 μl/min over the sensor chip.Kinetic binding data were double referenced according to Myszka, 1999and fitted by BIA evaluation software 4.1 (Biacore AB) to a two-phasereaction model. Kinetic rate constants were approximated globally,maximal responses were fitted locally, and the bulk response was set tozero.

In order to quantify DC8E8's affinity for each of the tested tauproteins, the association equilibrium binding constants (K_(A)) weredetermined for DC8E8 binding to the four repeat tau protein isoform2N4R, three repeat tau protein isoform 2N3R, as well as to misdisorderedtauΔ(1-150;392-441)/4R and misdisordered tauΔ(1-150;392-441)/3R. All tauproteins used for SPR were prepared according to Example 1. The affinityof DC8E8 was highest for four repeat tauΔ(1-150;392-441)/4R, followed bythe full-length four repeat tau isoform 2N4R, then for three repeattauΔ(1-150;392-441)/3R, and lastly for the three repeat full-length tauisoform 2N3R (FIG. 9A, B). These results confirmed: (1) the specificityof DC8E8 for the misdisordered form of tau, and (2) the selectivity ofDC8E8 for misdisordered tau (i.e., disease or pathological tau) over thefull-length tau (i.e., normal or physiological tau).

Real time monitoring of binding events using SPR enabled the measurementof the kinetic rate of association (kON) and dissociation (kOFF) betweenDC8E8 and several tau proteins. DC8E8's binding kinetics revealed analtered conformation for misdisordered tauΔ(1-150;392-441)/4R andtauΔ(1-150;392-441)/3R, when compared to physiological 2N4R tau, whichis indicated by more easily accessible DC8E8 epitope(s) in themisdisorderd tau proteins. This is reflected by the faster binding andhigher kON for the misdisordered tau proteins compared to theirfull-length counterparts. Moreover, the presence of an extra bindingsite for DC8E8 on the four-repeat tau protein species resulted in a10-times slower dissociation of 4R tau species from the complex withDC8E8 and a corresponding 10-times lower kOFF (FIG. 10A, B; the dashedlines were interpolated from measured data by kinetic parametercalculations using a computer program BIAEvaluation v4.1).

Example 6 DC8E8 Recognizes all Developmental Stages of NeurofibrillaryDegeneration in Human Alzheimer's Disease Brain

Human brain tissue (on paraffin blocks) were obtained from theNetherlands brain bank. The blocks were cut on a microtome.Paraffin-sections (8 μm) of the hippocampus-entorhinal cortex fromAlzheimer's disease brain (Braak's stage VI) and non-demented control(Braak's stage I and III) were treated with cold (+4° C.) 99% formicacid for 1 min at room temperature (25° C.). The tissue sections wereincubated in blocking solution (5% BSA, 0.3% Triton X-100 in 50 nMTris-HCl) and then overnight with purified primary antibody DC8E8 (7.8mg/ml; prepared as described in Example 5), which was diluted 1:2,000 inblocking solution. Subsequently, the sections were incubated with abiotinylated secondary antibody (Vectastain Elite ABC Kit, VectorLaboratories) at room temperature for an hour and then reacted withavidin-biotin peroxidase-complex for 60 minutes (Vectastain Elite ABCKit, Vector Laboratories), both at room temperature (25° C.). Theimmunoreaction was visualized with peroxidase substrate kit (Vector VIP,Vector laboratories, Ca, USA) and counterstained with methyl green(Vector Laboratories). The sections were examined with an Olympus BX71microscope.

Monoclonal antibody DC8E8 discriminated between preclinical AD,clinically incipient AD, and fully developed final stage of AD.Immunohistochemical study showed that DC8E8 detected early stages (taumonomers, dimers) of pathological tau in human preclinical AD—Braak'sStage I. (FIG. 11A). The brain contains only a limited number ofneurofibrillary tangles (NFTs) in the entorhinal cortex and no NFTs inthe hippocampus (Braak's stage I). In the clinically incipient AD brain,where a few NFTs were found in the hippocampus (Braak's stage III), theDC8E8 mAb recognized both the stage of pathological tau oligomers(arrows) and the stage of pathological tau polymers (tangles) (FIG.11B). In fully developed Alzheimer's disease brain, where extensiveneurofibrillary degeneration is present, DC8E8 recognizes mainlypathological tau polymers in the form of neurofibrillary tangles,neuritic plaques, and neurotic threads (FIG. 11C). Thus, mAb DC8E8recognizes all developmental stages of neurofibrillary lesions in humanAlzheimer's disease brain tissue, including monomeric, dimeric,early-oligomeric stage (FIG. 11D1) and late-oligomeric pre-tangle stage(FIG. 11D2), as well as late developmental stages of pathological taupolymers—intracellular (FIG. 11D3) and extracellular neurofibrillarytangles (FIG. 11D4). This reactivity of mAb DC8E8 is thus useful forboth diagnostic and therapeutic applications of this antibody.

Example 7 DC8E8 Recognizes all Developmental Stages of NeurofibrillaryDegeneration in the Brain of Transgenic Rats SHR72, as Seen in HumanAlzheimer's Disease

The SHR24 Transgenic Rat Line:

this line expresses tauΔ(1-150;392-441)/3R, a protein described inInternational Patent Application PCT WO 2004/007547. Generation andcharacterization of this transgenic line has been described in Filipciket al., 2010. These transgenic rats develop progressive age-dependentneurofibrillary degeneration in the cortical brain areas.Neurofibrillary tangles (NFTs) satisfied several key histologicalcriteria used to identify neurofibrillary degeneration in humanAlzheimer's disease including argyrophilia, Congo red birefringence, andThioflavin S reactivity. Neurofibrillary tangles were also identifiedwith antibodies used to detect pathologic tau in the human brain,including DC11, which recognizes a disease tau conformation (Vechterovaet al. 2003; Kovacech et al. 2010), and antibodies that are specific forhyperphosphorylated forms of tau protein. Moreover, neurofibrillarydegeneration was characterized by extensive formation of sarkosylinsoluble tau protein complexes consisting of rat endogenous andtransgenic truncated tau species (Filipcik et al., 2010). The mostprominent histopathological feature of these transgenic rats isextensive neurofibrillary pathology—neurofibrillary tangles in thecortex. The median survival time of transgenic rats is 222.5 days(SD=43.56) and the longest survival period reaches 475 days (Filipcik etal., 2010).

The SHR72 Transgenic Rat Line:

These transgenic rats express human truncated tauΔ(1-150;392-441)/4Raccording to International Patent Application PCT WO 2004/007547) inseveral brain regions and spinal cord. Generation of this rat line wasdescribed by Zilka et al., 2006, and tau pathology was described inKoson et al., 2008. The most prominent histopathological feature ofthese transgenic rats is extensive neurofibrillary pathology, e.g.,neurofibrillary tangles. The appearance of NFTs satisfied severalhistological criteria used to identify neurofibrillary degeneration inhuman AD including argyrophilia, Congo red birefringence, and ThioflavinS reactivity. NFTs were also identified with antibodies used to detectpathologic tau in the human brain, including DC11, recognizing anabnormal tau conformation (see U.S. Pat. No. 7,446,180), and antibodiesthat are specific for hyperphosphorylated forms of tau protein.Moreover, neurofibrillary degeneration was characterized by extensiveformation of sarcosyl-insoluble tau protein complexes consisting of ratendogenous and human truncated tau species. In a heterozygote line ofthis model the most extensive neurofibrillary pathology was observed inthe brainstem and spinal cord (Zilka et al., 2006). The transgeneexpression levels, the NFT load, and the rats' life span have beenpreviously determined. The median survival time for the transgenic rats(line SHR72) was 222.5 days (SD=24.48) (Koson et al., 2008).

Transgenic rat lines SHR24 (express tauΔ(1-150;392-441)/3R) and SHR72(express tauΔ(1-150;392-441)/4R) develop extensive neurofibrillarydegeneration in the brain and spinal cord. Transgenic rat line SHR24displays severe neurodegeneration in the isocortex, brainstem and spinalcord, while SHR72 transgenic rats develop NFT mainly in the brainstemand spinal cord but not in cortex. Progression of sensorimotor andneurological impairment is similar in both transgenic lines; howeverSHR72 transgenic rats show shorter lifespan.

In the transgenic rat studies presented in this application, hemizygoustransgenic rats were used (SHR24 and SHR72). All rats were housed understandard laboratory conditions with free access to water and food andwere kept under diurnal lighting conditions (12 hour light/dark cycleswith light starting at 7:00 a.m.). Efforts were made to minimize thenumber of rats utilized and to limit their discomfort, pain, andsuffering.

Immunohistochemistry of rat brain tissue with DC8E8:

Transgenic rats (7 months old) were perfused transcardially with PBS for1 min under deep anesthesia followed by perfusion with 100 ml of 4%paraformaldehyde (pH 7.4). After perfusion, the head was cut off and thebrain was quickly removed. The brain was cut sagittally into twoequal-sized hemispheres using disposable scalpel blades. The braintissues were post-fixed in 4% paraformaldehyde, embedded in paraffin,and cut into sections on a microtome. Immunohistochemistry andhistopathology were done on 8 μm paraffin-embedded tissue sections.Tissue sections were pre-treated for 20 min with an antigen unmaskingsolution (Vector laboratories, CA, USA) and for 1 min with cold (+4° C.)90% formic acid (Applichem, Germany), at room temperature (25° C.).After blocking, the sections were incubated overnight with purifiedmonoclonal antibody DC8E8 (7.8 mg/ml) that was diluted 1:2000 inblocking solution (5% bovine serum albumin, 0.3% Triton X 100 in 50 nMTris-HCl). After washing, the sections were incubated with abiotinylated secondary antibody (Vectastain Elite ABC Kit, VectorLaboratories) at room temperature for an hour, and then reacted with anavidin-biotin peroxidase-complex solution for 60 minutes (VectastainElite ABC Kit, Vector Laboratories), at room temperature (25° C.). Theimmunoreaction was visualized with a peroxidase substrate kit (VectorVIP, Vector laboratories, Ca, USA), and the sections were counterstainedwith methyl green (Vector Laboratories). Sections were examined with anOlympus BX71 microscope.

In the transgenic rat brain (SHR72), mAb DC8E8 recognized the diseasestage of pathological tau oligomers (arrows) and the disease stage ofpathological tau polymers (tangles) (FIG. 12A). Moreover, DC8E8 reactedwith misfolded tau that is located in the axonal fibers. In age-matchedcontrol rat brains the antibody did not stain neuronal soma or axonalprocesses (FIG. 12B).

As in human Alzheimer's disease brain (see supra), mAb DC8E8 recognizedall developmental stages of neurofibrillary lesions in the brain ofSHR72 transgenic rats, including diseased monomeric, dimeric, andearly-oligomeric stage (FIG. 12C) and late-oligomeric pre tangle stagetau (FIG. 12D), as well as late developmental stages of pathological taupolymers—intracellular (FIG. 12E) and extracellular neurofibrillarytangles (FIG. 12F).

DC8E8 also recognized neurofibrillary tangles in the brain of transgenicrats expressing tauΔ(1-150;392-441)/3R) (SHR24, FIG. 13A; SHR72, FIG.13B).

Example 8 DC8E8 Recognizes Both Soluble Misdisordered Tau and InsolubleTau Species in Human Alzheimer's Disease and in Brains of Tau TransgenicRats

Soluble tau and insoluble tau complexes were isolated either from humanAD brains or from disease tau transgenic rat brains (SHR24 and SHR72lines described in Example 7), using the sarkosyl method (Greenberg andDavies, 1990). For protein extraction, frozen human AD brain tissues(allocortex, samples of Braak stages V and VI obtained form theNetherlands brain bank) and tissues from transgenic SHR24 rats(isocortex, 10, 12 and 14 months old) and from transgenic SHR72 rats(brain stem, 7.5 months old) were homogenized in 10 volumes of coldextraction buffer (10 mM Tris pH 7.4, 0.8 M NaCl, 1 mM EGTA, and 10%sucrose). The homogenates were centrifuged for 20 min at 20,000×g and 50μl of the supernates were used for the analysis of soluble tau.

To prepare sarkosyl-insoluble tau, the remaining supernates weresupplemented with N-lauroylsarcosine (SIGMA) to a final concentration of1% and incubated for 1 h at room temperature, while shaking. Aftercentrifugation at 100,000×g for 1 h, the resulting supernates werediscarded, and the pellets comprise the sarkosyl-insoluble tau fraction.

Soluble tau and sarkosyl-insoluble tau fractions were analyzed byimmunoblotting. Soluble tau fractions were diluted with an equal volumeof 2x SDS-sample loading buffer (with β-mercaptoethanol) (Laemmli, 1970)and 15 μg of proteins were loaded per lane. For sarkosyl-insoluble taufractions, the pellets were dissolved in 1×SDS-sample loading buffer, in1/50 volume of the soluble fraction used for the preparation of theinsoluble tau fraction. Then, equal volumes of soluble tau andsarkosyl-insoluble tau fractions were used for immunoblot analysis,which corresponded to 15 μg of total protein in the soluble fraction(see Filipcik et al. 2010). Samples were heated at 95° C. for 5 min,loaded onto 5-20% gradient SDS polyacrylamide gels, and electrophoresedin a Tris-glycine-SDS buffer system for 40 minutes at 25 mA. Proteinswere transferred to a polyvinylidene fluoride (PVDF) membrane (1 h at150 mA in 10 mM CAPS, pH 12). After the transfer, the membranes wereblocked in 5% non-fat dry milk in phosphate-buffered-saline (PBS; 136.89mM NaCl, 2.7 mM KCl, 8.09 mM Na₂HPO₄, 1.47 mM KH₂PO₄) for 1 h at roomtemperature, and then incubated for 1 h with DC8E8 hybridoma culturesupernate, diluted 1:1 with TBST-milk (20 mM Tris-HCl, pH 7.4, 150 mMNaCl, 0.1% Tween 20, 5% non-fat dried milk), followed by three washeswith large volumes of PBS. The membranes were incubated (1 h at roomtemperature) with HRP-conjugated goat anti-mouse Ig (DAKO, Denmark),diluted 1:4,000 with PBS, as a secondary antibody. This incubation wasfollowed by washing (three times) with 0.2% Igepal CA-630 (SIGMA) inPBS. The blots were developed with SuperSignal West PicoChemiluminescent Substrate (Pierce, U.S.A), and the protein signalsdetected using a LAS3000 imaging system (FUJI Photo Film Co., Japan).The chemiluminescence signal intensities were quantified using AIDA(Advanced Image Data Analyzer, Raytest, Straubenhardt, Germany)software.

DC8E8 recognized both soluble human tauΔ(1-150;392-441)/3R andphysiological rat tau isoforms in SHR24 transgenic rats (FIG. 14A).Moreover, DC8E8 recognized tauΔ(1-150;392-441)/3R and pathological rattau proteins in the sarcosyl-insoluble tau fractions from SHR24 ratbrains (FIG. 14B). Importantly, DC8E8 strongly recognized pathologicalhuman tau proteins in the sarcosyl-insoluble tau fraction in the humanAD brains (Braak stages V and VI, FIGS. 14B and 14C). DC8E8 recognizedboth soluble human tauΔ(1-150;392-441)/4R and full-length(physiological) rat tau isoforms in SHR72 transgenic rats (FIG. 14D).Significantly, DC8E8 specifically recognized pathologicaltauΔ(1-150;392-441)/4R and pathological forms of rat tau in thesarcosyl-insoluble tau fractions from SHR72 rat brains (FIG. 14D).

Example 9 DC8E8 Inhibits Pathological Tau-Tau Interactions

The Tau Fibrillization Assay.

An in vitro tau fibrillization assay was used to determine whether DC8E8had an inhibitory effect on pathological tau-tau interactions. The assayis based on an intrinsic property of tau proteins, namely their abilityto undergo a conformational change upon interaction with polyanions,such as the sulfated glycosaminoglycan heparin. This alteredconformation on one tau molecule further leads to its pathologicalinteractions with another tau molecule, stabilization of the tau-taucomplex through formation of cross-6 sheet structures in the microtubulebinding regions of the interacting tau molecules, and, lastly, formationof Alzheimer's disease-like paired helical filaments (PHFs) (Skrabana etal., 2006). The formation of the beta-sheet-rich structures can bedetected by fluorescent dyes, like Thioflavin T.

The assay to measure the effect of DC8E8 on pathological tau-tauinteractions was setup in PBS (filtered through a 0.2 μm filter)containing: 20 μM (final concentration) of either one of the testedrecombinant tau proteins (tauΔ(1-150; 392-441)/4R ortauΔ(1-296;392-441)/4R), purified as described in Example 1; 5 μMheparin (Heparin sodium salt from porcine intestinal mucosa, 150 IU/mg,dry basis, from SIGMA); and 12.5 μM (final concentration) Thioflavin T.Each reaction (80 μl final volume) was incubated for 20 h at 37° C. insealed black solid polystyrene plates (384 wells, Greiner BioOne).Thioflavin T fluorescence was measured using a fluorescence reader(Fluoroskan Ascent FL (Labsystems)), with an excitation wavelength of450 nm, emission at 510 nm, and 200 ms measurement time. For determiningthe inhibitory activity of mAb DC8E8 on pathological tau-tauinteractions, purified DC8E8 (Example 5) was added to the reaction mixat 20 μM final concentration, prior to the incubation at 37° C. Twoantibodies were used as controls: DC51 (recognizing an envelope proteinof the rabies virus; Macikova et al., 1992) and DC11 (recognizingcertain truncated conformationally altered forms of tau, U.S. Pat. No.7,446,180).

The amount of conformationally altered and fibrilized tau was measuredby Thioflavin T fluorescence in the absence (“Ctrl”) and in the presence(“DC8E8”) of DC8E8 (FIGS. 15A and 15B). mAb DC8E8, added at 20 μM finalconcentration, prevented the pathological conformational change andfibrillization of both misdisordered tau proteins, reducing the amountof fibrillized pathological tau forms to less than 5% and 16% fortauΔ(1-150;392-441)/4R and tauΔ(1-296;392-441)/4R, respectively. Thisinhibitory activity of DC8E8 was statistically significant when analyzedby a non-parametric t-test (“DC8E8”, p<0.001 and p p<0.01 in FIGS. 15Aand 15B, respectively). An irrelevant antibody, Rab50 (Macikova et al.,1992), which does not bind tau, did not prevent the conformationalchange of tau, resulting in unaltered Thioflavin T fluorescence(“Rab50”). The antibody DC11, which recognizes certain pathologicallyaltered conformations of tau (Vechterova et al., 2003 and U.S. Pat. No.7,446,180), further promoted the formation of fibrilized tau; thiseffect can reflect a stabilization of the pathological conformation oftau required for the abnormal tau-tau interaction and fibril formationby DC11.

DC8E8 also inhibited the formation of tau dimers, trimers, and oligomersby misdisordered tauΔ(1-296;392-441/4R) (FIG. 16). Recombinant tauΔ(1-296;392-441)/4R was incubated for 1, 4, and 20 h either in thepresence or in the absence of DC8E8 as described above for thefibrillization assay. At the time points indicated, the reaction wasstopped by addition of SDS-sample loading buffer. For protein analysis,10 μl of each fibrillization reaction was loaded onto 5-20% gradient SDSpolyacrylamide gels and electrophoresed in Tris-glycine-SDS buffersystem for 40 minutes at 25 mA. After protein transfer to PVDF membranes(1 h at 150 mA in 10 mM CAPS, pH 12), the membranes were blocked in 5%non-fat dry milk in PBS for 1 h at room temperature, and then incubatedfor 1 h with HRP-conjugated DC25 (Skrabana et al. (2006) diluted 1:1,000in PBS, followed by three washes with large volumes of PBS. The blotswere developed with SuperSignal West Pico Chemiluminescent Substrate(Pierce, U.S.A), and the chemiluminescence signals detected using aLAS3000 imaging system (FUJI Photo Film Co., Japan). Thechemiluminescence signal intensities were quantified using AIDA(Advanced Image Data Analyzer, Raytest, Straubenhardt, Germany)software.

These results reveal that one or more of the four binding sites thatDC8E8 recognizes/binds to in human tau is involved in monomer tauconformational changes, tau fibrillization, and in the formation of tauaggregates (dimers, trimers, and other oligomers). In other words, oneor more of the four regions of tau encompassed by residues267-KHQPGGG-273 (SEQ ID NO: 98) (1^(st) repeat domain of tau protein),298-KHVPGGG-304 (SEQ ID NO: 99) (2^(nd) repeat domain of tau protein),329-HHKPGGG-335 (SEQ ID NO: 100) (3^(rd) repeat domain of tau protein),and 361-THVPGGG-367 (SEQ ID NO: 101) (4^(th) repeat domain of tauprotein) promotes and/or is involved in tau fibrillization and formationof tau aggregates (dimers, trimers, and other oligomers).

Example 10 Dc8E8 Mediates Uptake and Degradation of Misdisordered Tau

Mouse BV2 microglia cells were treated in 6-well plates for differenttime periods with either 1 μM recombinant tauΔ(1-150;392-441)/4R alone,or with a mixture/complex of tauΔ(1-150;392-441)/4R and DC8E8. Themedium was collected and the cells were washed first with PBS, and thenfor 1 min with mild acid wash solution (0.5 M NaCl, 0.2 M acetic acid,pH 3). The washed cells were then lysed in TTL buffer (20 mM Tris pH7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Triton X-100, 50 mM NaF, 1mM Na₃VO₄, Roche—protease inhibitor complete) and quickly frozen inliquid nitrogen. The resulting cell extracts were analyzed on 12%SDS-PAGE gel and Western blot as described previously (Koson et al.,2008). Briefly, the proteins were transferred onto nitrocellulosemembranes (Millipore, Billerica, Mass., USA) and stained with Ponceau Sto confirm uniform protein transfer, and then the membranes were probedwith DC25 hybridoma culture supernate recognizing tau residues 347-353,and referred to as a pan-tau antibody (Axon Neuroscience, Vienna,Austria). Western blotting with an anti-GADPH antibody (1:1,000, Abcam)was used as a protein loading control. Incubation with DC25 primaryantibody was followed by washes and incubation with a polyclonal goatanti-mouse IgG secondary antibody, which was HRP-conjugated (1:3,000;Dako, Glostrup, Denmark). The blots were developed with SuperSignal WestPico Chemiluminescent Substrate (Pierce, U.S.A.), and thechemiluminescence signals detected using a LAS3000 imaging system (FUJIPhoto Film Co., Japan).

TauΔ(1-150;392-441)/4R was added to cultures of mouse BV2 cells at theconcentration of 1 μM either alone or with 1 μM mAb DC8E8, as describedin the previous paragraph. After incubation for 2, 4, 6, and 12 hours,the cellular proteins were extracted, and the levels of internalized tauwere analyzed by Western blotting. Pan-tau antibody DC25 showed thepresence of misdisordered tau inside the microglial cells. The westernblot profile revealed that the degradation of misdisordered tau wasfaster in the presence of DC8E8 (FIG. 17A).

The DC8E8 antibody itself was also found present inside the BV2 cells.FIG. 17A. Moreover, DC8E8 reduced the load of soluble misdisordered tauin the cell medium, which can reflect the activation of theextracellular proteolytic machinery (FIG. 17B).

Example 11 DC8E8 is Stable at 37° C.

DC8E8 (purified as described in Example 5) was diluted to concentrationof 2 mg/ml in PBS and aliquots (100 μl) were incubated at 37° C. Atvarious 1-month intervals, aliquots were frozen at −20° C. An aliquot ofDC8E8 (2 mg/ml) kept stored at −20° C. throughout the duration of theexperiment was used as a “control.” After 4 months (when all sampleswere collected) analysis of DC8E8 activity (binding to recombinanttauΔ(1-150;392-441)/4R as a solid phase), hence shelf-life stability at37° C., was done by ELISA, as described in Example 2. Each DC8E8 aliquotwas diluted 2,000-fold (i.e., 2,000× or 1:2,000), 4,000×, 8,000×,16,000×, 32,000×, 64,000×,128,000×, 256,000×, and 512,000×. DC8E8 wasactive (as measured by its ability to bind tauΔ(1-150;392-441)/4R) andthus stable even after 4 months of incubation at the 37° C., as comparedto the “control” (FIG. 18).

Example 12 DC8E8 is Capable of Binding to and Immunoprecipitating BothSoluble and Insoluble Tau from Human AD Brain and from the Brain ofSHR72 Rats, Under Native Ex Vivo-Like Conditions

Sarkosyl-insoluble misfolded tau proteins were biochemically isolatedeither from human AD brains or from tau transgenic rat brains (lineSHR72 described in Example 7) using the sarkosyl method (Greenberg andDavies, 1990). For protein extraction, unfixed frozen human AD brain(transentorhinal cortex, Braak stage V, obtained from the NetherlandsBrain Bank, Netherlands) and SHR72 transgenic rat (isocortex, 7.5 monthsold animals) tissues were homogenized in 10 volumes of ice-coldextraction buffer [10 mM Tris pH 7.4, 0.8 M NaCl, 1 mM EGTA, and 10%sucrose (supplemented with 50 mM NaF, 1 mM Na₃VO₄, and the cocktail ofprotease inhibitors Complete® without EDTA (from Roche)]. Thehomogenates were centrifuged for 20 min at 20,000×g to removemembraneous material. To prepare sarkosyl-insoluble tau fractions, thesupernates were supplemented with N-lauroylsarcosine (SIGMA) to a finalconcentration of 1% and incubated for 1 h at room temperature, whileshaking. After centrifugation at 100,000×g for 1 h, the resultingsupernates were discarded and the pellets were washed once in 3 ml ofphosphate-buffered saline (PBS, 8.09 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 136.89mM NaCl, 2.7 mM (KCl)). The pellets, which represent the brain proteinfraction enriched in oligomeric and polymeric misfolded tau species(i.e. the disease tau proteins), were then re-suspended in 1 ml of PBS(supplemented with 50 mM NaF, 1 mM Na₃VO₄ and the cocktail of proteaseinhibitors Complete® without EDTA (Roche)) by sonication for 2 minuteson ice using a Bandelin Sonopuls HD2200/UW2200 equipped with a MS72probe, at 20% duty cycle with the output set at 20% (BandelinElectronic, Germany).

The resulting suspensions (both from human AD brain and from the brainof rat AD model), enriched in the disease tau proteins, were split intotwo 500 μl portions and each portion received 25 μg of one of twopurified antibodies: either DC8E8 or control antibody Rab50 (recognizingan envelope protein of the rabies virus; Macikova et al., 1992). Thesuspensions were incubated with the antibodies with head-over-tailrotation at 6° C. for 2 hours. In order to isolate the formedantibody-disease tau complexes, 50 μl of 50% suspension of Protein G MagSepharose beads (GE Healthcare) equilibrated in PBS were added into eachsuspension reaction, which were further incubated at 6° C. for 1 hour.The beads with bound antibody-tau complexes were harvested and washedthree times with PBS (supplemented with 50 mM NaF, 1 mM Na₃VO₄, 0.02%IGEPAL CA-630 (SIGMA) and the cocktail of protease inhibitors Complete®without EDTA (Roche)). The bound antibody complexes were eluted from thebeads by three separate 5-min incubations in 100 μl of 200 mM formicacid pH 2.7. The 100 μl eluates were pooled, lyophilized, the proteinsdissolved in SDS-PAGE sample loading buffer (Laemmli, 1970), separatedon 12% SDS-PAGE gels, transferred onto nitrocellulose membranes, and thetau proteins detected by incubation with the pan-tau antibody DC25(epitope 347-353 of 2N4R tau, Axon Neuroscience, Vienna, Austria),HRP-conjugated as above (Kementec, Denmark). Incubation (1 h at roomtemperature) was followed by washing (three times) with 0.2% IgepalCA-630 (SIGMA) in PBS. The blots were developed with a SuperSignal WestPico Chemiluminescent Substrate system (Pierce, U.S.A) and the signalsdetected using aLAS3000 imaging system (FUJI Photo Film Co., Japan).

DC8E8 recognizes, targets, and binds all forms of the disease tauproteins: oligomeric and polymeric misfolded tau species present in thebrain of a patient with Alzheimer's disease (FIG. 19A). Lane 1 in FIG.19A shows biochemically extracted pathological tau species from human ADbrain. Lane 3 shows tau species recognized, bound by, and isolated byimmunoprecipitation with DC8E8. The pattern ofDC8E8-bound/immunoprecipitated disease tau species was that of thebiochemically extracted tau proteins. These results show efficient exvivo recognition by DC8E8 of the pathological tau species in the humanbrain, i.e., in extracts where the proteins are in vivo-like, unmodifiedform, showing that DC8E8 has useful therapeutic properties, being ableto target the disease tau species in vivo. The control antibody Rab50(“Mock antibody”, lane 2) does not recognize any of the tau proteinspresent in the brain extract, confirming that binding of DC8E8 to tauproteins is specific.

FIG. 19B shows the amount of mock antibody (Rab50) and DC8E8 (lanes 2and 3, respectively) used for the immunopurification of tau proteins.The positions of the heavy and light chains of DC8E8 are also marked inlane 3 of FIG. 19A. The presence of the higher amount of antibody chainsdistorts the pattern of disease tau.

DC8E8 also recognizes and targets all forms of misfolded (diseased) tauin the brain of the SHR72 rat model of Alzheimer's disease. FIG. 20A,lane 1 shows biochemically extracted disease tau species from the brainof the transgenic rats. Incubation of DC8E8 antibody with the transgenicrat brain extract allowed immunopurification of disease tau speciespresent in the brain (FIG. 20A, lane 3). The DC8E8 purified tau speciesshowed a pattern identical to that of biochemically isolated tauproteins (FIG. 20A, lane 1), which confirms that DC8E8 recognizes andbinds all pathological tau species present in the transgenic rat brain.The slight distortion of the tau proteins banding pattern is caused bythe presence of DC8E8 antibody heavy and light chains (marked in FIG.20A, lane 3). Mock antibody Rab50 (FIG. 20A, lane 2) did not bind any ofthe tau proteins.

FIG. 20B shows the amount of mock antibody (Rab50) and DC8E8 (lanes 2and 3, respectively) used for the immunopurification of tau proteinsfrom the brain extract of rats of the transgenic model of Alzheimer'sdisease. The positions of the heavy and light chains of DC8E8 are alsomarked in lane 3 of FIG. 20A. The presence of the higher amount of theantibody chains slightly distorted the pattern of disease tau.

Example 13 DC8E8 Monoclonal Antibody Removes Pathological Tau from theBrain of Transgenic Rats SHR72

Hybridoma cells producing either DC8E8 or Rab50 (a negative controlantibody, recognizing virus rabbies) were cultivated in DMEM containing10% NHS and 1% glutamine. The cells were counted in a Bürker countingchamber. Cell suspensions containing 500,000 cells per milliliter werespun down at 100×g for 5 min and the pellets were resuspended in 1 ml ofPBS. The cell suspensions were spun down again at 100×g for 5 min, andthe pellets resuspended in 5 μl of PBS.

Transgenic rats SHR72 (6 months old) were used for these experiments (3rats per group). At least one hour before surgery, an immunosuppressivedrug—Sandimmun (15 mg/kg)—was applied to the rats subcutaneously.Transgenic rats were anaesthetized by mixture of Tiletamine-Zolazepam(100 mg/ml)/Xylazine (20 mg/ml) in 3:5 ratio, injectedintraperitoneally. Dosing of anesthetics was as follows: Zoletil (30mg/kg) and Xylariem (10 mg/kg). The heads of the anesthetized rats werefixed in a stereotactic apparatus (David Kopf Instruments, Tujunga, CA,USA) by placing fixating arms into the ear canals of each animal. Holeswere drilled on each animal's head, using the surgical drill, accordingto chosen stereotactic coordinates (lateral 5 mm; anterior-posterior 4mm; dorsoventral −5 mm, relative to bregma). The hybridoma cellsuspensions, producing either DC8E8 (10⁵ cells) or Rab50 (10⁵ cells),were bilaterally injected into the fimbria of hippocampi of the rats'brains. Shortly after operation, Ketonal (5 mg/kg) was administerintramuscularly. Sandimmun (15 mg/kg) was applied subcutaneously for 8days after application. Enroxil (20 mg/kg/24 h) was administered indrinking water for 10 days.

Two weeks after the surgical procedure, the rats were anaesthetized by amixture of Zoletil (30 mg/kg) and Xylariem (10 mg/kg). After 2-5minutes, rats were mounted on a dissecting stage, and their abdominalcavity was opened. Before perfusion the blood was collected for analysisof tau and antibody levels in the blood serum. A perfusion needle wasplaced into the left heart ventricle, and the rats were perfused withPBS for 2 minutes using a peristaltic pump (Type pp1-05, Zalimp,speed—10×, degree 7-22 ml/1 min of perfusion liquid). Each rat wasdecapitated, its skull was opened by paean, and the brain (with part ofthe spinal cord) carefully removed. Brains were cut sagitally into twoparts; the right side was fixed in 4% PFA (4° C.) overnight. The leftside was cut and the brainstem and two cortical areas were quicklyfrozen in liquid nitrogen.

The amount of DC8E8 antibody in the serum of the treated animals wasdetermined by ELISA, as described below in Example 19 using tauΔ(1-150;392-441)4R as a solid phase. The serum of each animal (A, B, C) wasserially diluted from 100× to 12,800× (FIG. 21A). The serumconcentration of DC8E8 antibody was determined using purified DC8E8 as astandard. DC8E8 reached concentrations of 466, 200, and 273 ng/ml intreated animals (A, B, C, of the DC8E8 treatment group, respectively).

The concentration of tau in the sera of each treated animal was alsodetermined. This was done using a Innotest hTAU ELISA kit (Innogenetics,Belgium), according to the manufacturer's protocol. Treatment with DC8E8caused transport of the antibody-tau complexes into the blood, where taureached the average concentration of 350 μg/ml. This effect of DC8E8helps to eliminate pathological tau proteins from the brain. On theother hand, no tau proteins were detected in the sera of the animalstreated with mock antibody (Rab50), which recognizes the envelopeprotein of the rabies virus (Macikova et al., 1992). The graph showsmeans with standard errors of the mean (SEM) (FIG. 21B).

The fixed brain tissues were embedded in paraffin and cut on amicrotome. Immunohistochemistry was done on 8 μm paraffin-embeddedsections. Tissue sections were pretreated for 20 minutes with boilingantigen unmasking solution (Vector Laboratories, Burlingame, Calif.,USA) and for 1 minute with 85% formic acid (Applichem, Darmstadt,Germany). After blocking, the tissue sections were incubated with mAbDC8E8 overnight, followed by washes and incubation (1 hour, at roomtemperature) with biotinylated secondary antibody (Vectastain Elite ABCKit, Vector Laboratories). After washing, the sections were reacted withan avidin-biotin peroxidase-complex (Vectastain Elite ABC Kit, VectorLaboratories) for 60 minutes at room temperature. The immunoreaction wasvisualized with peroxidase substrate kit VIP (Vector VIP, VectorLaboratories, Burlingame, Calif., USA). The superior olivary complex wasused for quantification of DC8E8 intraneuronal signal. The total signalwas quantified in individual motor neurons, at least 15 neurons persection. The image analysis was done using AIDA (Advanced Image DataAnalyzer, Raytest, Straubenhardt, Germany) software. The counting wasdone on sections of all treated animals and then statistically evaluatedusing nonparametric Mann-Whitney test.

The quantification (FIG. 22B) of the amount of pathological tau in thesuperior olivary complex of sections from the mock-treated (FIG. 22A,right panel) and DC8E8-treated (FIG. 22A, left panel) animals showed areduction in the amount of pathological tau in the tested brain area inall three animals treated with DC8E8 compared to mock-treated animals(p<0.0001) (FIGS. 22A and 22B).

Example 14 Mapping of the Residues within the DC8E8 Combining Site thatInfluence DC8E8's Recognition of Tau's Therapeutic Epitopes

a) Cloning of the Single Chain DC8E8 Antibody and Mutagenesis of theAntibody Combining Site.

A functional single chain version (scDC8E8v) of the full-length DC8E8mAb was prepared to help map the amino acid residues of mAb DC8E8essential for the recognition of tau proteins. This was done using cDNAsof DC8E8's light and heavy chains prepared as described in Example 3.

The variable regions of DC8E8 were further amplified by PCR usingcloning primers bearing restriction sites for NcoI (forward primer:5′-ATATTACCATGGACATTGTGATGTCACAG-3′ (SEQ ID NO: 155)) and XhoI (reverseprimer: 5′-ATATTATTCTCGAGGGAGACGGTGACTGAGGT-3′ (SEQ ID NO: 156))restriction enzymes and oligonucleotide linker sequences (VH-LINK-F:5′-GGCGGCGGCGGCTCCGGTGGTGGTGGTTCCATGCAGGTCCAATTGCAGCAG-3′ (SEQ ID NO:157); VL-LINK-R: 5′-GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCCCGTTTGATGTCCAGCTTGGTGCC-3′ (SEQ ID NO: 158)), which were used to join heavyand light chain variable regions (the linker sequence was designedaccording to Krebber et al. 1997). After digestion of the isolated PCRproducts (using NcoI and XhoI enzymes) and purification via agarose gelelectrophoresis, the fragments were cloned into a NcoI- andXhoI-digested pET22b plasmid, using T4-DNA-Ligase (Fermentas, Germany).The bacterial strain DH5α was used for the amplification of theresulting plasmid and the correct insert position in the selected cloneswas verified by restriction enzyme analysis. The correctness of thesequence of the resulting single-chain DC8E8 construct (scDC8E8v) wasverified by DNA sequencing using a 3130 Genetic Analyzer (AppliedBiosystems, USA).

b) Identification, by Alanine Scanning Mutagenesis, of Residues in DC8E8Combining Site that Influence DC8E8's Recognition of Pathological Tau.

For the identification of residues that influence DC8E8 binding to tau,Ala scanning mutagenesis was done on select sites (in bold andunderlined, see below) of scDC8E8v. The amino acids selected for Alasubstitution were identified on the basis of the work of MacCallum et al(J. Mol. Biol. 1996). The mutagenesis of scDC8E8v was done by overlapextension method using mutant oligonucleotides in PCR by standardprocedures described in “Molecular Cloning: A Laboratory manual” bySambrook and Russell (2001).

The mutant single chains are listed below, relative to each originalDC8E8 sequence. The mutated residues are in bold and underlined. Thenames of the mutant single chains consist of the number of theconstruct, variable heavy or light chain acronym (VH or VL), thesingle-letter code of the original amino acid in DC8E8 followed by itsposition in the light and heavy chain sequence SEQ ID Nos.141 and No.138, respectively, and then A for the substituting alanine. For example,mutant 1-VL-N31A corresponds to single chain mutant number 1, where thevariable light chain was mutated at position 31 (relative to DC8E8), byreplacing Asparagine 31 (N31) with Alanine (A)):

DC8E8 Light chain variable region (CDRs areunderlined): [SEQ ID No. 141]: 1        10        20        30        40DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQS50        60        70        80        90PKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSF  100     110YLRTFGGGTKLDIK Mutations in CDR1 [SEQ ID No. 117] (the mutatedresidues are in bold and underlined): (SEQ ID NO: 117) QSLL N S R TRKN Y(original DC8E8 sequence of CDR1) [SEQ ID NO. 247] 1-VL-N31A[SEQ ID NO. 248] 2-VL-R33A [SEQ ID NO. 249] 3-VL-Y38AMutation in Framework region FR2 preceding CDR2: (SEQ ID NO: 160)LAWYQQKPGQSPKLLI Y (original DC8E8 sequence of framework region FR2)[SEQ ID NO. 252] 4-VL-Y55A Mutation in CDR2 [SEQ ID No. 118]:(SEQ ID NO: 118) W AS  (original DC8E8 sequence of CDR2)[SEQ ID NO. 253] 5-VL-W56A Mutations in CDR3 [SEQ ID No. 119]:(SEQ ID NO: 119) KQSFYLR T (original DC8E8 sequence of CDR3)[SEQ ID NO. 254] 6-VL-K95A [SEQ ID NO. 255] 7-VL-Q96A [SEQ ID NO. 256]8-VL-S97A [SEQ ID NO. 257] 9-VL-F98A [SEQ ID NO. 258] 10-VL-Y99A[SEQ ID NO. 259] 11-VL-L100A [SEQ ID NO. 260] 12-VL-R101ADC8E8 Heavy chain variable region (CDRs areunderlined): [SEQ ID No. 138]: 1        10        20        30        40QVQLQQSGPELVKPGTSVKMPCKASGYIFTDYVISWVKQRTGQGLEWIG50        60        70        80        90EIFPRSGSTYYNEKFKGKATLTADKSSNTAYMQLSSVTSEDSAVYFCAR 100      110DYYGTSFAMDYWGQGTSVTVSS Mutations in CDR1 [SEQ ID No. 120] (the mutatedresidues are in bold and underlined): (SEQ ID NO: 120) GYIFTD YV IS(original DC8E8 sequence of CDR1) [SEQ ID NO. 261] 14-VH-Y32A[SEQ ID NO. 262] 15-VH-V33AMutation in Framework region FR2 preceding CDR2: (SEQ ID NO: 162)WVKQRTGQGLEWIG E (original DC8E8 sequence of framework region FR2)[SEQ ID NO. 263] 16-VH-E50A Mutations in CDR2 [SEQ ID No. 121]:(SEQ ID NO: 121) I F PRSG S T (original DC8E8 sequence of CDR2)[SEQ ID NO. 264] 17-VH-F52A [SEQ ID NO. 265] 18-VH-S57AMutations in CDR3 [SEQ ID No. 122]: (SEQ ID NO: 122) AR DYYG TSFAMDY(original DC8E8 sequence of CDR3) [SEQ ID NO. 266] 19-VH-D99A[SEQ ID NO. 267] 20-VH-Y100A [SEQ ID NO. 268] 21-VH-Y101A[SEQ ID NO. 269] 22-VH-G102A

Analytical and Preparative Expression of Recombinant DC8E8 AntibodyVariants:

The pET22b-scDC8E8v DNA constructs of wild type antibody and its mutants(all of which are His-tagged) were transformed into E. coli cells of theproduction strain BL21(DE3). The resulting clones were first verifiedfor the production of recombinant scDC8E8v. Individual colonies obtainedafter transformation were inoculated into 2 ml of LB medium supplementedwith 100 μg/ml of ampicillin and grown at 37° C. for 5 hours withconstant agitation (Sambrook and Russell 2001). A 100 μl aliquot of eachculture was removed, mixed with ⅔ of the volume of 100% glycerol andstored frozen at −80° C. for later use. The expression of therecombinant protein was induced by the addition ofisopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of1 mM and incubation continued for an additional 3 hours. The cells werecollected by centrifugation in a benchtop centrifuge at 4° C. for 1 minat 10,000×g, the supernate was discarded, the cell pellet wasresuspended in 1×SDS-sample buffer (Laemmli 1970) and boiled for 5minutes. The samples were centrifuged for 5 minutes at 10,000×g andsupernates (bacterial lysates) analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). The separated proteins were visualized bystaining with Coomassie Brilliant Blue R250 (SIGMA).

For preparative expression of each scDC8E8v antibody (native andmutant), bacterial cells containing the respective expression plasmidswere cultivated and induced as described in “Molecular Cloning: ALaboratory manual” by Sambrook and Russell (2001). Transformed cellswere grown at 37° C. in 100-500 ml of LB medium with 100 μg/mlampicillin at 230 rpm. When absorbance of the culture at 600 nm reached0.8-1.0, IPTG was added to a final concentration of 1 mM to induce theexpression of scDC8E8v. After further incubation at 37° C. for 3 hours,the cells were collected by centrifugation at 3,000 g for 15 min at 4°C. The cell pellet was resuspended in 10 ml of lysis buffer (50 mM PIPESpH 6.9, 50 mM NaCl, 0.1 mM PMSF) and sonicated on ice 6 times for 30 swith 30 s pauses using a TT-13 tip (50% duty cycle, 50 W power output,Sonopuls HD 2200, Bandelin, Germany). The lysate was clarified bycentrifugation (21,000×g for 15 min at 4° C.). Lysates were filteredthrough a 0.45 μm membrane filter and stored at −80° C. until use. Forexamination of the successful induction and the level of expression, 1ml of the induced culture was collected, cells harvested bycentrifugation as above, resuspended in 100 μl of 1×SDS sample buffer,boiled for 5 minutes at 95° C. and then analyzed by SDS-PAGE (FIG. 23A).The cytoplasmic lysate was used for the binding/detection of tauproteins (by western blot analysis) and for the SPR determination of therecombinant antibodies' affinity for tau, both of which are measures ofthe activity of the native and mutant scDC8E8v antibodies.

Example 15 Recombinant scFV Fragment of Monoclonal Antibody DC8E8(scDC8E8V) Recognizes Pathological Tau (TauΔ(1-150;392-441)/4R)

For determination of the binding properties of scDC8E8v to pathologicalforms of tau, the protein lysate from bacteria expressing scDC8E8v wasdiluted 16-fold into TBS-T buffer (20 mM Tris pH 7.4, 150 mM NaCl, 0.1%Tween 20) and was used to overlay the nitrocellulose membrane containing500, 250, and 125 ng of recombinant truncated tau protein(tauΔ(1-150;392-441)/4R) and recombinant C-terminally truncated tauprotein (tauΔ228-441) (Ponceau S staining of the proteins, FIG. 23B).Bound scDC8E8v was detected by immunoblotting with an anti-6×hisTag (SEQID NO: 163) antibody (No. A00174; GenScript, Piscataway, N.J., USA) andvisualized with anti-rabbit HRP-conjugated antibody (DAKO Cytomation,Denmark) (FIG. 23C). The blots were developed with SuperSignal West PicoChemiluminescent Substrate (Pierce, U.S.A) detected using anLAS3000imaging system (FUJI Photo Film Co., Japan).

Western blot analysis showed that recombinant single chain antibody(scDC8E8v) detects truncated tau [tauΔ(1-150;392-441)/4R]. The bindingis specific, since scDC8E8v does not recognize the control truncated tauprotein tauΔ228-441, which does not contain any of the four therapeuticepitopes recognized by intact DC8E8 (FIG. 23C lane 4). Probing thenitrocellulose membrane containing the tau proteins (loaded as shown inFIG. 23B) with lysate from control bacteria not expressing scDC8E8v didnot produce any signal (FIG. 23D).

Example 16 Recombinant scFV Fragment of Monoclonal Antibody DC8E8(scDC8E8v) Exhibits Tau Binding Properties Similar to the DC8E8Antibody—Selectively Recognizes Pathological Tau(TauΔ(1-150;392-441)/4R) and Significantly Discriminates it fromPhysiological, Native Tau (Tau2N4R)

To quantify the tau binding properties of recombinant single chainantibody scDC8E8v, kinetic affinity determinations were done by SPR(Biacore 3000, Biacore, Sweden) to measure scDC8E8v's binding to thepathological truncated tau (tauΔ(1-150;392-441)/4R) (FIG. 24A) and tothe normal human four repeat tau isoform 2N4R (FIG. 24B). To this end,11,000 RU of the rabbit polyclonal anti-His tag antibody (No. A00174;GenScript, Piscataway, N.J., USA) were immobilized on a CM5 sensor chip.All experiments were done at 25° C. in PBS pH 7.4 with 0.005% of P20(PBS-P) as the running buffer. Recombinant His-tagged scDC8E8v wascaptured in the analytical flow cell to reach an immobilization level of60 RU, and known concentrations of each tau protein, or of a PBS-Pcontrol sample, were injected at a flow rate of 100 μl/min over thesensor chip. Kinetic binding data were double referenced and fitted bythe BIA evaluation software 4.1 (Biacore AB) to a 1:1 interaction model.Kinetic rate constants were approximated globally, maximal responseswere fitted locally, and bulk response was set to zero.

The kinetic measurements showed a higher association rate constant and alower dissociation rate constant of scDC8E8v toward pathological ADtauΔ(1-150;392-441)/4R than toward normal tau 2N4R (FIG. 24C).Consequently, the affinity of scDC8E8v for truncated AD tau is higher(higher value of equilibrium association constant K_(A)) than for thefull-length tau isoform 2N4R. These measurements confirmed that thesingle chain version of DC8E8 antibody (scDC8E8v) shows a bindingpreference for conformationally altered, pathological tau proteins, likethe parental full-length DC8E8 antibody. Recombinant scDC8E8v is thussuitable for identification of amino acid residues within the DC8E8antibody combining site responsible for its binding properties

Example 17 Identification of Residues in the scDC8E8v Combining Sitethat Influence scDC8E8v/DC8E8's Recognition of Pathological Tau Epitopes

Several amino-acid residues that influence the DC8E8-antigen interactionhave been determined by alanine scanning mutagenesis of the residues ofscDC8E8v combining site. The potential antigen-contacting residues inthe light and heavy chains were identified on the basis of the work ofMacCallum et al (J. Mol. Biol. 1996) and mutated to alanine, asdescribed in Example 14. The mutant versions of scDC8E8v weresubsequently expressed in BL21 E. coli strain (FIG. 25A, single chainproteins are indicated by asterisks). The binding properties of themutated scDC8E8v were analyzed by western blotting (FIG. 25B-C). FIG.25B shows PonceauS staining of nitrocellulose membranes containingvarious amounts of truncated tauΔ(1-150;392-441)/4R protein. Identicalmembranes were used for detection of the truncated tau by the variousmutant single chain antibodies (FIG. 25C).

Based on these results, the amino acid residues in the variable regionof DC8E8 were classified into three main categories, as follows:

Category 1: The residues listed in this category (in bold) contributedthe most for binding of scDC8E8v to pathological truncated AD tau(tauΔ(1-150;392-441)/4R). Mutation of any one of these residues toalanine most prevented recognition of DC8E8 epitopes on tau protein.

Category 1 Residues in DC8E8 Light Chain:

-   -   Asparagine at the position 31 in CDRL1 [SEQ ID No. 117]    -   Tyrosine at the position 38 in CDRL1 [SEQ ID No. 117]    -   Serine at the position 97 in CDRL3 [SEQ ID No. 119]

Category 1 Residues in DC8E8 Heavy Chain:

-   -   Glutamic acid at the position 50 in FRH2 preceding CDRH2 [SEQ ID        No. 121]    -   Tyrosine at the position 101 in CDRH3 [SEQ ID No. 122]

Category 2: The residues listed in this category contribute to bindingof scDC8E8v to pathological tau (tauΔ(1-150;392-441)/4R). Mutation ofany one of these residues to alanine reduces reactivity of scDC8E8vtoward truncated AD tau (tauΔ(1-150;392-441)/4R), but prevents it to alesser degree than mutations to Category 1 residues:

Category 2 residues in DC8E8 light chain:

-   -   Tyrosine at the position 55 in framework region FRL2 preceding        CDRL2 [SEQ ID No. 118]    -   Lysine at the position 95 in CDRL3 [SEQ ID No. 119]

Category 2 Residues in DC8E8 Heavy Chain:

-   -   Serine at the position 57 in CDRH2 [SEQ ID No. 121]    -   Tyrosine at the position 100 in CDRH3 [SEQ ID No. 122]    -   Glycine at the position 102 in CDRH3 [SEQ ID No. 122]

Category 3: The residues listed in this last category contribute theleast to binding of scDC8E8v to pathological AD tau(tauΔ(1-150;392-441)/4R). The mutation of these residues to alanine doesnot change the reactivity of scDC8E8v toward truncated tau(tauΔ(1-150;392-441)/4R):

Category 3 Residues in the Light Chain:

-   -   Arginine at the position 33 in CDRL1 [SEQ ID No. 117]    -   Tryptophane at the position 56 in CDRL2 [SEQ ID No. 118]    -   Glutamine at the position 96 in CDRL3 [SEQ ID No. 119]    -   Phenylalanine at the position 98 in CDRL3 [SEQ ID No. 119]    -   Tyrosine at the position 99 in CDRL3 [SEQ ID No. 119]    -   Leucine at the position 100 in CDRL3 [SEQ ID No. 119]    -   Arginine at the position 101 in CDRL3 [SEQ ID No. 119]

Category 3 Residues in the Heavy Chain:

-   -   Tyrosine at the position 32 in CDRH1 [SEQ ID No. 120]    -   Valine at the position 33 in CDRH1 [SEQ ID No. 121]    -   Phenylalanine at the position 52 in CDRH2 [SEQ ID No. 121]    -   Aspartic acid at the position 99 in CDRH3 [SEQ ID No. 122]

Example 18 Active Vaccination with Therapeutic Tau Epitopes: Preparationof Immunogens for Vaccines Based on One of the DC8E8 Binding Epitopes onTau and Vaccine Administration

a. Peptides:

Peptide immunogens consisting of synthetic fragments of human tauprotein 2N4R were synthesized by Antagene, Inc. (Sunnyvale, Calif.) andEZBiolab, USA, with purity higher than 95%. Each peptide sequence wasdesigned to encompass at least one of the sequences believed to beinvolved and/or promote tau fibrillization/aggregation (novel target“therapeutic epitopes”), and which epitopes were identified above asbinding sites to DC8E8 and by the assays described in Examples 1-11. SeeFIG. 26A. These four sequences are also referred to herein as“therapeutic epitopes” (see below). These epitopes represent tau-tauinteraction motifs (aggregation epitopes), which are important for taufibrillization/PHF assembly. Therefore, targeting these strategictherapeutic epitopes can lead to the successful treatment of AD andrelated tauopathies. Furthermore, usage of such a specific anti-tautherapy would prove safer than widely aimed therapies relying onrandomly selected tau epitopes, because targeting some tau epitopes hasbeen thought to provoke an autoimmune response and potentially aggravatethe disease (Furlan et al., 2003; Gruden et al., 2004).

Accordingly, to further determine the therapeutic potential of tau-basedimmunotherapy, several sets of tau peptides were designated for use asimmunogens. All of the residues refer to those of full-length tau 2N4R,which has 441 amino acid residues. A first set of immunogens was made upof SEQ ID NO:1 tau 251-PDLKNVKSKIGSTENLKHQPGGGKVQIINK-280; SEQ ID NO:2tau 256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285; SEQ ID NO:3 tau259-KIGSTENLKHQPGGGKVQIINKKLDLSNVQ-288; SEQ ID NO:4 tau 275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304; SEQ ID NO:5 tau 201-GSPGTPGSRSRTPSLPTPPTREPKKVAWR-230; SEQ ID NO:6 tau 379-RENAKAKTDHGAEIVYKSPWSGDTSPRHL-408; SEQ ID NO:7 tau181-TPPSSGEPPKSGDRSGYSSPGSPGTPGSRS-210; SEQ ID NO:8 tau300-VPGGGSVQIVYKPVDLSK-317; and SEQ ID NO:108 tau 294-KDNIKHVPGGGS-305.Some of these sequences carry phoshorylated epitopes previously found inAD tau pathology. Thus, tau peptide SEQ ID NO:5 contains phosphorylatedthreonine at position 217, tau peptide SEQ ID NO:6 containsphosphorylated serine residues at position 396 and 404, and tau peptideSEQ ID NO:7 contains phosphorylated serine at position 202 and threonineat position 205. Tau peptide SEQ ID NO:2 was synthesized inunphosphorylated and phosphorylated form. The phosphorylated formcontains phosphorylated serine residue at position 262.

Within this set, one subset of immunogens (tau peptides SEQ ID NOs:1-4and 108) encompass one of the therapeutic epitopes represented either bySEQ ID NO:98, (tau 267-KHQPGGG-273) or SEQ ID NO:99 (tau298-KHVPGGG-304). The another subset of tau peptides (SEQ ID NOs:5-7)encompass phosphorylated sites, found in Alzheimer's disease and othertauopathies. Tau peptide SEQ ID NO:8 does not carry any of the mentionedepitopes and was used as a control. (FIG. 26)

The second set of peptides was made up of SEQ ID NOs: 9 through 97,which represent overlapping sequences spanning residues 244-390 of humantau 2N4R. Each of these peptides comprises one of the tau therapeuticepitope sequences in a different tau-based environment. In other words,in each subset, each of the epitopes #1 through #4 is surrounded bydifferent tau residues on both its N- and C-terminus. (FIG. 26).

b. Conjugation for Use as a Vaccine:

Tau peptides SEQ ID NOs: 2, 4, 7, and 108 were conjugated to keyholelimpet hemocyanin (KLH) via a cysteine link.

To this end, tau peptides SEQ ID NO: 2, 4, 7, and 108 were synthetizedas cysteinated peptides with an extra N-terminally located cysteineresidue with the aim to obtain oriented attachment of the peptide on thesurface of the KLH protein. Peptides were coupled to the KLH carrier viabifunctional cross-linker N-[γ-maleimidobutyryloxy]succinimide ester(GMBS). To prepare the conjugation reaction, 20 mg of KLH (Calbiochem)were dissolved in conjugation buffer (PBS with 0.9 M NaCl, 10 mM EDTA)to a concentration of 10 mg/ml by gentle mixing for 10 minutes. Forpreparation of maleimide-activated KLH, 2 mg of active bi-functionalcross-linker GMBS were dissolved in 50 μl of anhydrous dimethylformamideand mixed with 2 ml of KLH solution for 1 hour at room temperature.Subsequently, un-reacted GMBS was removed on a 5 ml HiTrap Desaltingcolumn (GE Healthcare) equilibrated in conjugation buffer. Conjugationswere carried out at a 1:1 ratio of peptide to maleimide-activated KLH(w/w, 20 mg of peptide) for 2 h at room temperature (25° C.). Theresulting conjugates were dialyzed against a 100-fold excess of PBS,with four dialysis buffer changes to remove unconjugated peptide. Afterdialysis, the conjugates were centrifuged at 21,000×g for 15 min at 2°C. Completeness of conjugation was confirmed by the absence of freepeptide in the dialysis buffer, measured using LC-MS/MS. The conjugateswere aliquoted and stored at −20° C. until used.

c. Vaccine Preparation:

To prepare immunization doses with Aluminum/Alum adjuvant AdjuPhos(Brenntag Biosector, Denmark), 200 μg of each respective tau peptideconjugate (dissolved in 150 μl of PBS) were emulsified at a 1:1(vol/vol) ratio with AdjuPhos adjuvant, in a final dose volume of 300μl. Each suspension/emulsion was incubated with rotation at 4° C.overnight to allow the peptide to adsorb onto the aluminum phosphateparticles.

To prepare immunization doses with Complete Freund's adjuvant, 200 μg ofeach respective tau peptide conjugate (dissolved in 150 μl of PBS) wasemulsified 1:1 (vol/vol) with Complete Freund's adjuvant, in a finaldose volume of 300 μl. For subsequent booster doses, the immunogen wasprepared similarly, but was emulsified with Incomplete Freund'sadjuvant.

d. Vaccine Administration:

Prepared vaccine doses were injected into tau transgenic rats carrying ahuman truncated tau transgene (SHR72 line described above in Example 7,expressing tauΔ(1-150;392-441)/4R (Zilka et al., 2006). The ratsreceived the first subcutaneous injection of 200 μg of immunogen in afinal volume of 300 μl at 2 months of age, followed by the secondinjection three weeks later, and thereafter on a monthly schedule. Thecontrol transgenic rats received the adjuvant mixed 1:1 with PBS. Theevaluation of the efficacy of the immunotherapy is described in Example19.

Isolation and Purification of Fetal Rat Tau, for Use as InternalStandard in the Quantitative Analysis of Insoluble Tau inVaccine-Treated Rats:

Fetal rat tau purification was done essentially as described inIvanovova et al., 2008, using 1% perchloric acid. Brain tissue obtainedfrom 1-7 day old rat pups was homogenized in ice-cold 1% perchloric acid(1.5 g tissue per 5 ml of perchloric acid, PCA) and allowed to stand onice for 20 min. The homogenate was spun at 15,000×g for 20 min, and theclear supernate was concentrated and simultaneously the buffer waschanged to washing buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween20) using an Amicon Ultra Centrifugal Filter device (Millipore). Thefiltered PCA brain extracts, containing about 10 mg of total proteins,were loaded at a flow rate of 0.2 ml/min onto a Poly-Prep column C10/10(GE Healthcare) packed with Sepharose carrying immobilized pan-tau mAbDC25 (see above). Unbound proteins were washed off with 10-15 ml washingbuffer until the absorbance of the eluting fractions (at 280 nm) becamestable. Fetal tau, bound to mAb DC25, was eluted with 0.1 M glycine, pH2.6. Eluted 0.5 ml fractions were immediately neutralized with 50 μl of1 M Tris-HCl, pH 9, and assayed by SDS-PAGE. Fractions containing fetaltau were concentrated using Amicon Ultra Centrifugal Filter devices(Millipore) with simultaneous buffer exchange to PBS. Fetal tau purifiedby affinity chromatography was precipitated according to Chen et al.,(2005) by addition of four volumes of ice-cold acetone containing 10%trichloroacetic acid. The mixture was incubated at −20° C. for 2 hoursand centrifuged at 15,000×g for 20 min at 2° C. The supernate wasdiscarded and the precipitate was resuspended in 1 ml of ice-coldacetone, allowed to stand on ice for 20 min and again centrifuged asabove. The resulting pellet was dried at room temperature and dissolvedin a volume of PBS equal to that volume before precipitation.

Example 19 General Methods of Evaluating Tau Peptide Vaccines inTransgenic Rat Models of Alzheimer's Disease

Active vaccines were evaluated by the following approaches: (a)biochemically, by evaluating the effect of their administration on thelevels of phosphorylated forms of insoluble tau using immunoblotanalysis of rat brain samples with phosphorylation-specific monoclonalantibodies AT270, DC209, DC217, and AT8, and total amount of insolubletau using the pan-tau monoclonal antibody DC25; (b) neurobehaviorally,using a NeuroScale evaluation; (c) immunohistochemically, including NFTquantification; and (d) by analysis of the induced antibody response.Passive vaccines can also be evaluated pre-clinically by one or more ofthese approaches, among others.

(a). Biochemical Analysis:

Insoluble tau fractions were prepared using the sarkosyl method(Greenberg and Davies, 1990), from the brain stems of transgenic (SHR72)rats immunized with test peptides, as well as from mock-treated SHR72rats (injected with adjuvant only (control)), as described in Example 8.Sarkosyl-insoluble tau samples were analyzed by immunoblotting usingvarious monoclonal antibodies, as described below. The results of thisanalysis for immunogenic peptides SEQ ID NOs 1-8 and 108 are summarizedin FIG. 26B. The levels of insoluble tau have been shown to correlatewith progression of tau pathology in SHR72 transgenic rats. Koson etal., 2008.

Immunoblot Analysis:

Samples of sarkosyl-insoluble tau fractions were dissolved in 1× sodiumdodecyl sulfate (SDS) sample loading buffer (Laemmli, 1970) in 1/50volume of the soluble fraction (see Filipcik et al. 2010) and heated at95° C. for 5 min. 6 μl of each were then loaded onto 5-20% gradient SDSpolyacrylamide gels and electrophoresed in Tris-glycine-SDS buffersystem for 40 minutes at 25 mA. Proteins were transferred to PVDFmembranes (1 h at 150 mA in 10 mM CAPS, pH 12). After the transfer, themembranes were blocked in 5% non-fat dry milk in PBS for 1 h at roomtemperature and then incubated for 1 h with primary (tau-specific)monoclonal antibodies (see below for more detailed descriptions of eachantibody), followed by three washes with a large volume of PBS. Afterwashes, HRP-conjugated goat anti-mouse Ig (DAKO, Denmark) diluted 1:4000with PBS was used as a secondary antibody. Incubation (1 h at roomtemperature) was followed by washing (three times) with 0.2% Igepal inPBS. The blots were developed with SuperSignal West PicoChemiluminescent Substrate (Pierce, U.S.A.) and the signal detectedusing a LAS3000 imaging system (FUJI Photo Film Co., Japan). The signalintensities were quantified using AIDA software (Advanced Image DataAnalyzer, Raytest, Straubenhardt, Germany). Fetal tau (0.6 μg/lane) wasused as an internal standard for quantification analysis.

Monoclonal antibodies AT8 and AT270 were purchased from Innogenetics(Belgium) and both were used for immunoblot analysis. AT8 was also usedfor immunohistochemistry. AT8 recognizes phosphorylated serine 202 andthreonine 205 located in the proline-rich domain of tau, whichcontributes to a major part of tau's microtubule-binding affinity. AT270recognizes phosphorylated threonine 181 located also in the proline-richdomain. Both antibodies bind soluble and insoluble tau. Thephosphorylation-specific monoclonal antibodies DC209 (recognizes pT231),DC217 (recognizes pT217) used in this study were prepared by AxonNeuroscience, GmbH (Vienna, Austria). Pan-tau antibody DC25 (AxonNeuroscience, GmbH) recognizes an epitope in the fourthmicrotubule-binding repeat repeat at the C-terminus of tau protein(347-353) in all forms of soluble and insoluble tau. DC25 antibodyrecognizes tau proteins independent of their phosphorylation level.

(b). Neurobehavioral Evaluation:

Neurobehavioral responses in the rats were assessed 10 days after thefinal vaccine dose using the NeuroScale, which is a battery ofbehavioral tests originally designed for transgenic rats expressinghuman truncated tau protein (Korenova et al., 2009). The NeuroScale iscomposed of sensorimotor (beam walking test), neuromuscular (prehensiletraction test), and neurological tasks (placing, righting, postural,pinna, startle, and hindlimb escape extension reflex), enriched withbasic observational assessments.

The general observation involved assessment of posture and limbfunctions; the neurological examination included the basic reflexresponse, all graded on a 1-point scale (normal response 0; delayed orincomplete response 1 point). Assessment of the hind-limb escapeextension reflex was graded on a 3-point scale (normal response 0-1;deficit 2-3 points).

For the beam walking test, three sorts of traversing segments were used(3×3 cm, 4×2 cm, and a round beam of 3.5 cm diameter). The maximumnumber of points was 10 (5 points for latency+5 points for hind-limbslips on one beam type). The lower the score obtained from the beamwalking test, the better the sensorimotor-coordination ability of thetested animal. The sum of points possible to achieve was 30.

For the prehensile traction test, a rat was allowed to grasp, with itsforepaws, a horizontal steel wire (3 mm in diameter) suspended 76 cmabove a padded surface. Latency to fall from the wire was measured. Themaximal number of points awardable was 5, reflecting serious impairmentof neuromuscular functionality and muscular weakness. The longer thelatency to fall, the lower the score obtained from the task, reflectingforelimb muscular strength and the agility of the tested animal.

The NeuroScale score was calculated from the scores obtained in theindividual tests. A maximum total score of 49 points was possible byadding the contribution of the observational assessment, theneurological examination, the three series of beam walking test and theprehensile traction test. The more severe the neurobehavioralimpairment, the higher the NeuroScale score.

(c). Immunohistochemistry:

For the collection of brain samples, the rats were perfusedtranscardially with PBS for 2 minutes under deep anesthesia. Afterperfusion, the rat brains were removed and cut sagittally into twoequal-sized hemispheres. The brainstem and the cerebellum of the righthemisphere were collected for biochemical analyses. The left hemispherewas fixed with 4% paraformaldehyde overnight at 4° C., followed by atreatment with 25% sucrose for 48 hours to provide cryoprotection. Thematerial was then frozen in cold 2-methylbutane (−42° C.) for 30 s andsectioned on a cryomicrotome. Sagittal sections (40 μm) were cut in acryostat at −18° C. Free-floating sections were used forimmunohistochemical studies.

Immunohistochemical staining was done on frozen brain sections oftreated and control rats. Koson et al., 2008, have shown that the numberof NFT in the brain of SHR72 transgenic rats correlates with the time ofdemise of the animals (i.e., the more NFTs, the earlier the animal'sdemise). In order to analyze neurofibrillary changes in the rat brains,sagittal sections of the brain were prepared. Free floating tissuesections were treated with cold (+4° C.) 80% formic acid for 30 s atroom temperature (25° C.). Brain sections were incubated for 20 minutesat room temperature in PBS containing 0.3% Triton X-100 and 1% H₂O₂,followed by a 30-minute incubation in blocking solution (PBS containing0.3% Triton X-100, 1% horse serum), followed by overnight incubation at4° C. with either purified AT8 antibody (0.2 μg/ml in blocking buffer)or hybridoma culture supernantant DC217 (1:100 in blocking buffer). Bothantibodies showed similar immunostaining pattern in the brain stem oftransgenic rats. After washing, the sections were immunostained usingthe standard avidin biotin peroxidase method (ABC Elite, VectorLaboratories, Burlingame, Calif.). The reaction product was visualizedusing an avidin-biotin system and Vector VIP as a chromogen (VectorLaboratories). Sections were then examined with an Olympus BX 51microscope.

(d). Antibody Response:

The transgenic rats were bled before the commencement of the study(i.e., prior to the first injection) and two weeks after the lastinjection. The antibody response to the administered immunogen/vaccinewas determined by ELISA using serially diluted plasma samples. Peptideimmunogens, recombinant tau protein tauΔ(1-150;392-441)/4R, andrecombinant full-length tau isoform 2N4R, were separately coated onto 96well plates (IWAKI, Japan) at a concentration of 10 μg/ml in PBSovernight at 37° C. After blocking with 1% nonfat dried milk in PBS, theplates were washed with PBS-0.05% Tween 20 and incubated with 50 μl/wellof serial plasma dilutions (1:200-1:128,000 in blocking buffer) for 1 hrat 37° C. After incubation and washing, peroxidase-conjugated secondaryantibody (rabbit anti-rat Ig, DAKO, Denmark) was diluted 1:1000 andapplied to the wells (50 μl/well) for 1 hr at 37° C. The reaction wasdeveloped with o-phenylenediamine in a peroxidase substrate solution(0.1 M phosphate buffer) and stopped with 50 μl 2M H₂SO₄. Absorbance at492 nm was measured using a Multiscan MCC/340 ELISA reader (Labsystems).Absorbance readings of at least twice the value of the negative controlswere considered positive.

Affinity measurements were performed using SPR, as described above inExample 5. Briefly, experiments were performed at 25° C. inphosphate-buffered saline pH 7.4 with 0.005% of P20 (PBS-P) as therunning buffer. 3000 RU (response units) of polyclonal anti-mouseantibody (No. Z 0420; DakoCytomation, Glostrup, Denmark) were coupled ata concentration of 5 μg/ml (prepared by 40-times dilution of 200 μg/ml0.5×PBS stock in 10 mM sodium acetate buffer pH 4.5) via primary amines,simultaneously in two flow cells, one of which was used as a referencein the measurement. In each analysis cycle, 1000-fold diluted sera werecaptured in the analytical flow cell to reach an immobilization level˜850 RU, which approached saturation. For the K_(A) determinations, aswell as for the determination of kinetic rate constants, 100 nMsolutions of tau immunogenic peptides or tau proteins were injected at aflow rate of 100 μl/min over the sensor chip. PBS-P injection was usedfor background signal subtraction in the double referencing procedure(Myszka J Mol Rec 1999). Kinetic data were fitted by a BIA evaluationsoftware 4.1 (Biacore AB) to a 1:1 reaction model. Kinetic rateconstants were approximated globally, maximal responses were fittedlocally, and the bulk response was set to zero.

Example 20 Tau Peptide Vaccines Comprising at Least One of FourTherapeutic Epitope Sequences are Beneficial in Transgenic Rats thatModel Human Alzheimer's Disease

a. SEQ ID NO:1 tau 251-PDLKNVKSKIGSTENLKHQPGGGKVQIINK-280. Transgenicrats (SHR72) were immunized with tau peptide SEQ ID NO:1 formulated withAdju-phos adjuvant.

The conversion of tau from soluble to sarkosyl-insoluble pathologicforms is seen as an important step in the development of tau pathologyand appears to depend on several factors, such as the concentration oftau, the truncation of tau, and the extent of tau phosphorylation(Alonso et al. 2001; Koson et al. 2008; Kovacech and Novak 2010). Theresults of immunoblot quantitative analysis of insoluble tau in thesarkosyl insoluble brain fractions harvested from the group of ratsimmunized with tau peptide SEQ ID NO:1 and from the control group areshown in FIG. 26C. The vaccination reduced the amount of insoluble tauin rats immunized with tau251-280 (SEQ ID NO.1) compared to controlrats, which received adjuvant alone (FIG. 26C). This reduction wasobserved both for overall levels of insoluble tau (as assessed by theDC25 pan-tau antibody, recognizing residues 347-353) as well as for allother tested AD-relevant tau epitopes (surrogate markers of AD)described in FIGS. 26B and 26C and known to be present insarkosyl-insoluble fractions. Indeed, immunization with tau peptide SEQID NO:1 induced a statistically significant (p<0.001) reduction ininsoluble tau, which was observed at the total insoluble tau level (71%)using pan-tau monoclonal antibody DC25 (FIG. 26 B,C). Analysis withDC217 (pThr217), AT270 (pThr181), revealed a trend towards a reductionin the levels of phosphorylation at Thr217 (42%) and pThr181 (58%) ofinsoluble tau in the immunized rats compared to the control rats. Aweaker treatment effect (11%) was observed at the insoluble tau proteinphosphoepitope pThr231 (FIG. 26B). These results suggest that thevaccine activated a mechanism responsible for the inhibition of tauaggregation and/or a mechanism involved in decreasing the levels of tauproteins prone to tau-tau interaction.

Rats treated with the tau peptide immunogen SEQ ID NO:1 showedstatistically significant decreased escape latencies in the beam walkingtest (* p=0.045) compared to control rats (FIG. 27A). Similarly, thenumber of hind-limb slips was reduced in the vaccinated group comparedto controls; however, this difference was marginally statisticallysignificant (p=0.059, FIG. 27B). The NeuroScale score (described inExample 19, above) was calculated from the values obtained in the beamwalking tests, prehensile traction tests and neurological examinations(basic reflexes, hind-limb escape extension reflex). The immunizationimproved the NeuroScale score of the rats treated with peptide SEQ IDNO:1 compared to the control group, but this improvement was notstatistically significant (p=0.065, FIG. 27C). The total NeuroScalescore confirmed the neurobehavioral improvement of treated rats comparedto untreated rats. All statistical data were obtained using thenonparametric Mann-Whitney U-test.

The neurobehavioral parameters correlated with the insoluble tau levelsin the brain stem. Treated rats with low levels of insoluble tau showedlower escape latency and hind-limb slips compared to controls. Thesefindings indicate that the reduction of highly insoluble, misfolded tauleads to functional improvements in immunized rats, which could havetherapeutic value. Immunotherapy with tau peptide SEQ ID NO:1 resultedin improvement in neurobehavioral parameters of treated rats. Thiseffect followed the reduction in insoluble tau levels in the brains ofthe immunized rats. These findings indicate that lowering insoluble taulevels has therapeutic benefit.

The efficacy of the immunotherapy with tau peptide SEQ ID NO:1 wasfurther tested at the immunohistochemical level (FIG. 28).Neurofibrillary tangles (NFTs) were analyzed using anti-tau antibodiesAT8, DC217, which recognize phosphorylated epitopes on pathological tau,in the brainstems of treated and mock-treated control transgenic ratsSHR 72, which received adjuvant/PBS only. The number of NFTs wasdetermined using a semiquantitative method. Three quantitative levelswere assigned: 1) none or few neurofibrillary tangles (up to 3 in thebrain stem); 2) moderate (many NFTs mainly in the reticular formation ofbrain stem); and 3) severe (many NFTs in all areas of the brain stem).“Extensive” means moderate to severe stage of neurofibrillaryneurodegeneration. Immunohistochemical analysis showed a 50% reductionof neurofibrillary tangle load in vaccine-treated group of transgenicanimals.

The decrease of insoluble tau levels measured biochemically correlatedwith the immunohistochemical analysis results. The data fromimmunization with SEQ ID NO:1 show the treatment capacity of thispeptide, containing DC8E8 epitope No. 1.

b. SEQ ID NO:2 tau 256-VKSKIGSTENLKHQPGGGKVQIINK KLDLS-285.

Transgenic rats (SHR72) were immunized with tau peptide SEQ ID NO:2conjugated to the carrier KLH (as described above).

Immunoblot analysis revealed that the vaccine reduced the amount ofinsoluble tau in immunized rats compared with control rats that receivedadjuvant alone. FIG. 29 shows a reduction in all monitored epitopespresent in insoluble/aggregated tau. Quantitative analysis of pan-tauDC25 immunoreactivity revealed 41% reduction of insoluble tau(statistically significant, p<0.001) in immunized rats compared tocontrol rats that received adjuvant alone (FIG. 26B). Likewise,immunoreactivity of other antibodies recognizing phosphorylatedAD-specific epitopes on insoluble tau was reduced (FIG. 26B and FIG.29). Additionally, the treatment had an impact on the level of anothertau phosphoepitope, which is created by two phosphoresiduesSer202/Thr205 and which is known as a marker of AD pathology. Thevaccine induced a 80% reduction of this epitope in immunized transgenicrats, in comparison to non-treated animals. Similarly, the levels of thephosphorylated tau epitopes Thr217, Thr231, and Thr181, were decreasedby 72% (p<0.001), 64%, and 74% (p<0.01), respectively. These resultssuggest that the vaccine induced a mechanism responsible for theinhibition of tau aggregation and/or a mechanism lowering tau speciesprone to pathological tau-tau interaction.

The rats were subjected to behavioral analyses, and FIG. 30 showsresults obtained in the beam walking test (FIG. 30A), hind-limb slipstest (FIG. 30B) and neuroscale analysis (FIG. 30C, * p, 0.05). Apositive trend in beam walking escape latency was observed (p=0.096) inthe group of rats treated with the tau peptide SEQ ID NO:2. Theimmunization reduced the number of hind-limb slips in traverse beamtest; however, the difference was not statistically significant (p=0.25)in comparison to the control group. Motor impairment tests together withthe prehensile traction test and neurological examination weresummarized into Neuroscale score. FIG. 30 shows the statisticallysignificant improvement in Neuroscale score of the rats treated with taupeptide SEQ ID NO: 2 compared to the control group (* p=0.036). Ingeneral, the immunization with tau peptide SEQ ID NO: 2 improved theoverall motor performance. Improvement on neurobehavioral levelcorrelated with decrease in the amount of insoluble tau (AD-templatetau) in immunized animals compared to controls. These findings indicatethat lowering insoluble tau levels can have therapeutic benefit.

The efficacy of the immunotherapy with tau peptide SEQ ID NO:2 wasfurther tested at the immunohistochemical level (FIG. 31). For thispurpose, two different anti-tau antibodies were used (AT8, DC217), whichrecognize both phosphorylated soluble and insoluble tau. Neurofibrillarypathology, in these rats (SHR72), is localized mainly in the brainstemand spinal cord and partially in the cerebellum (data not shown). Thenumber of NFTs was determined using a semiquantitative method. Threequantitative levels were assigned: 1) none or few neurofibrillarytangles (up to 3 in the brain stem); 2) moderate (many NFTs mainly inthe reticular formation of brain stem); and 3) severe (many NFTs in allareas of the brain stem). “Extensive” means moderate to severe stage ofneurofibrillary neurodegeneration. Immunotherapy with peptide SEQ IDNO:2 decreased the number of transgenic rats with extensive NFTs in thebrainstem by almost 60% (FIG. 31).

These results demonstrate that vaccination with tau peptide SEQ ID NO:2shows several desirable vaccine outcomes: 1) reduction in insoluble tauin the brains of immunized transgenic rats at the biochemical level; 2)alleviation of sensorimotor deficits in the immunized trangenic rats atthe behavioral level; and 3) reduction in the numbers of neurofibrillarylesions at the immunohistochemical level.

SEQ ID NO:2 tau 256-VKSKIGSTENLKHQPGGGKVQIINKKLDLS-285 withphosphorylated Ser262. SEQ ID NO:2 containing phosphorylated serine atposition 262 was conjugated to KLH.

Immunoblot of rat brain sarkosyl-insoluble tau fractions with the DC25antibody revealed that the vaccine statistically significantly reducedthe total amount of insoluble tau by 46% (p<0.01) in the immunizedanimals compared to control transgenic rats that received adjuvant alone(FIG. 32 and FIG. 26B, DC25-based measurements (“347-353” data).Likewise, quantification of the signals of DC209 (pThr231), DC217(pThr217), AT8 (pSer202, pThr205) and AT270 (pT181) showed lower levelsof insoluble phosphorylated tau in the immunized group of animalscompared to controls (FIG. 26B). However, reduction of the levels ofphosphorylated insoluble tau at pThr217 (p<0.001; 73%), pThr231 (84%),pSer202/pThr205 (82%) and pThr181 (p<0.01; 82%) was more pronounced thanthe decrease observed in total insoluble tau. These changes in thelevels of phosphoepitopes involved in the AD tau misfolding cascade area useful indicator of the treatment effect. Reduction of AD-relevant tauepitopes shows that the vaccine activated a mechanism responsible forthe inhibition of tau aggregation and/or a mechanism decreasing levelsof tau prone to pathological interactions with endogenous tau.

The immunohistochemical profile (FIG. 33) shows that immunotherapy withthe phosphopeptide SEQ ID NO: 2 led to a decrease in the number oftransgenic rats with extensive NFTs in the brainstem. A 78% decrease inthe number of transgenic rats with extensive NFTs was observed in theimmunized group, when compared to the control non-immunized group. Theimmunization halted the development of the brain tau pathology inimmunized animals. A similar effect was obtained at the biochemicallevel, with a reduction of insoluble tau on the AD-relevant epitopesanalyzed (FIGS. 26B and 32).

These results demonstrate that vaccination with tau phosphopeptide SEQID NO:2/pSer262 provoked: 1) a reduction in insoluble tau in the brainsof immunized transgenic rats at the biochemical level; and 2) a positivetreatment effect on the numbers of neurofibrillary lesions at theimmunohistochemical level.

d. SEQ ID NO:3 tau 259-KIGSTENLKHQPGGGKVQIINKKLDLSNVQ-288. Transgenicrats (SHR72 line) were immunized with tau peptide SEQ ID NO:3 formulatedwith alum adjuvant (AdjuPhos).

FIG. 34 demonstrates that immunotherapy reduces insoluble tau in theimmunized rats compared to control rats that received adjuvant alone. Adecrease in the insoluble tau level was detected for all analyzed tauepitopes (i.e., for 347-353/DC25 antibody, pT217/DC217 antibody,pT231/DC209 antibody, pS202/pT205/AT8 antibody, and pT181/AT270antibody). The level of total insoluble tau was reduced by 40%, asrevealed by immunodetection with pan-tau antibody DC25. Similarly, thevaccine induced a 30% reduction in tau protein phosphorylated at pT217,as shown in FIG. 26B and FIG. 34. The treatment had a greater effect onthe levels of insoluble tau forms phosphorylated at Thr231 (63%), Thr181(74%), and Ser202/Thr205 (61%) in immunized rats, compared to thenon-immunized rats (FIG. 26B and FIG. 34). These findings show that areduction in insoluble tau can lead to additional alterations in thelevels of tau proteins leading to pathology and, thus, can havetherapeutic significance. The reduction of AD-relevant tau epitopesshows that the vaccine activated a mechanism responsible for theinhibition of tau aggregation and/or a mechanism decreasing levels oftau prone to pathological interactions with endogenous tau.

FIG. 35A-C shows results obtained by neurobehavioral evaluation. In thegroup of rats treated with the peptide SEQ ID NO: 3, a positive trend inbeam walking escape latency was observed; however, without astatistically significant difference between the peptide-treated andnon-treated/control rats (FIG. 35A; p=0.21). Similarly, treatment led toa decrease in numbers of hind limb slips in the group of immunized ratscompared to nontreated rats, but this effect was not statisticallysignificant (FIG. 35B, p=0.15). The total Neuroscale score confirmed theneurobehavioral improvement in rats treated with tau peptide SEQ ID NO:3 compared to untreated rats (p=0.11 in both cases). However, thedifference in Neuroscale score was not statistically significant(p=0.19). Immunotherapy with tau peptide SEQ ID NO: 3 resulted in bettersensorimotor coordination of the treated rats compared to control ratsin the beam walking test, as well as in the hind-limb slips test; theseresults were confirmed by the Neuroscale score.

Moreover, the immunohistochemical profile revealed that immunotherapywith the phosphopeptide SEQ ID NO: 3 lead to a 58% reduction of NFTs inthe brain stem of vaccine-treated animals (FIG. 36). These results showthat immunization with SEQ ID NO:3, which contains DC8E8 epitope No. 1,significantly reduced pathologic polymeric tau assembled into NFTs.Furthermore, this positive treatment effect was also shown at thebiochemical level, represented by a statistically significant reductionof insoluble pathological tau.

e. SEQ ID NO:4 tau 275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304.

Tau peptide SEQ ID NO:4 was conjugated to KLH and was used forimmunization with alum adjuvant (AdjuPhos).

FIG. 37 and FIG. 26B show that immunotherapy with SEQ ID NO:4 leads to areduction in the amount of insoluble tau in the immunized rats, comparedto control rats that received adjuvant alone. The data show that adecrease in the insoluble tau level was detected for all analyzed tauepitopes. Total insoluble tau levels were reduced by 63%, as revealed byimmunoreactivity with pan-tau antibody DC25. Similarly, the vaccineinduced reductions in tau protein phosphorylated at pThr217 (92%),Thr231 (95%), Thr181 (87%), and Ser202/Thr205 (95%), as shown in FIG.26B. It has been shown previously that the level of insoluble taucorrelates with the progress of tau pathology (Zilka et al., 2006). Thepresent results show that immunotherapy directed at the therapeutic tauepitope comprised within SEQ ID NO: 99 can decrease the levels ofinsoluble tau and retards the progress of tau pathology.

Complex motor impairment was measured by the set of standard motor testscombined with the neurological examination in the compositescore—Neuroscale. At 6.5 months of age, transgenic rats SHR72 treatedwith tau peptide SEQ ID NO:4 were subject to behavioral tests with aimto determine the effect of this immunotherapy. Rats treated with the taupeptide immunogen SEQ ID NO:4 showed decreased escape latencies in thebeam walking test, compared to the transgenic rats that receivedadjuvant alone (controls) (FIG. 38A). Similarly, positive results innumber of hind-limb slips was observed in the vaccinated group incomparison with transgenic treatment controls (FIG. 38B). The totalNeuroscale score was calculated from the data obtained in the beamwalking tests, prehensile traction tests, and neurological examinations(basic reflexes, hind-limb escape extension reflex). The immunizationimproved the Neuroscale score of the rats treated with peptide SEQ IDNO:4, compared to the control treatment group (FIG. 38C). The totalNeuroscale score confirmed the neurobehavioral improvement of treatedtransgenic rats when compared to untreated transgenic rats.

The efficacy of the immunotherapy with tau peptide SEQ ID NO:4 wasfurther tested at the immunohistochemical level (FIG. 39).Neurofibrillary tangles (NFTs) were analyzed using anti-tau antibodiesAT8 and DC217, which recognize phosphorylated epitopes on pathologicaltau, in the brain stems of vaccine-treated and adjuvant-treated(control) SHR72 rats. Brain tissue from animals which received adjuvantonly contained AT8- and DC217-positive NFTs in all areas of the brainstem, and mainly in the reticular formation of brain stem.Immunohistochemical analysis showed a 66% reduction of neurofibrillarypathology in the vaccine-treated transgenic rats SHR72 (FIG. 39).

The changes of insoluble tau levels in the brain stem of transgenic ratline SHR72 are a sensitive indicator of treatment effect. Insolubletotal tau, as well as phosphorylated tau levels (pathological monomers,dimers, oligomers and polymers), were both effectively reduced in thebrain stem by treatment with tau peptide SEQ ID NO:4 (63-95% reduction;FIG. 26B). The decrease of insoluble tau levels measured biochemicallycorrelated with the results obtained from immunohistochemical analysis.This analysis showed more than 60% reduction of neurofibrillarypathology in the brain stem of vaccine-injected transgenic rats (FIG.38). The data from immunization with SEQ ID NO:4, show the treatmentcapacity of this peptide, containing DC8E8 epitope No. 2.

f. SEQ ID NO:5 tau 201-GSPGTPGSRSRTPSLPTPPTREPKKVAVVR-230 carryingphosphorylated threonine at position 217. The tau peptide SEQ ID NO:5,phosphorylated at position of threonine 217, was administered to SHR72transgenic rats using alum adjuvant (AdjuPhos).

Immunoblot analysis showed that immunization with the phosphopeptide SEQID NO:5 did not affect the amount of total insoluble tau (detected withthe DC25 antibody) compared with control Tg rats that received adjuvantalone (FIG. 40 and FIG. 26B). However, analysis with antibodies DC209and AT270 revealed about 30% reduction in the phospho-tau pThr231 andpThr181 levels in the insoluble tau fraction (FIG. 26B). On the otherhand, treatment induced a moderate increase in insoluble tauphosphorylated at threonine 217 (11% increase) and tau carryingphosphosites Ser202/Thr205 (31% increase) compared to control transgenicrats. This finding would suggest an ambiguous or neutral vaccine effectof the immunization with phosphopeptide SEQ ID NO: 5 on the assessedAD-relevant markers, which peptide does not encompass any of the tauaggregation epitopes #1 through #4, identified above (Examples 1 through11) as therapeutic epitopes.

Neurobehavioral analyses of rats treated with phosphorylated tau peptideSEQ ID NO: 5 showed that there was no significant improvement inneurobehavioral functions in the treated group in beam walking test(FIG. 41A, p=0.19) or in the number of hind-limb slips when compared tothe control group (FIG. 41B). The total NeuroScale score (FIG. 41C)confirmed no neurobehavioral improvement of treated rats compared tomock-immunized rats (p=0.28).

Immunotherapy with tau phosphopeptide SEQ ID NO:5 did not reduce thenumber of transgenic rats (SHR72) with extensive NFTs in the brainstemwhen compared to control transgenic rats by immunohistochemistry (FIG.42).

These results show that vaccination with tau peptide SEQ ID NO: 5 pT217,which lacks all of the therapeutic epitopes (SEQ NO:98-101), does notresult in a statistically significant improvement of neurobehavioralfunction and only a reduction of approximately 11% in the number ofneurofibrillary lesions at the immunohistochemical level. No effect wasobserved on biochemical level with respect to a reduction of insolubletau, as assessed by the DC25 antibody.

g. SEQ ID NO:6 tau 379-RENAKAKTDHGAEIVYKSPVVSGDTSPRHL-408 CarryingPhosphorylated Serine Residues at Position 396 and 404.

Tau peptide phosphorylated at position of 396 and 404 was administeredto SHR72 using alum adjuvant (AdjuPhos). SEQ ID NO:6/pS396/pS404 lacksany of the therapeutic epitopes represented by SEQ NO:98-101, butcontains a phosphoepitope over-represented in AD brain tau proteins(Greenberg et al. 1992; Otvos et al. 1994).

In contrast to the decrease in the amount of sarkosyl-insoluble tauobserved with peptides SEQ ID NOs. 1-4, immunization with phosphorylatedpeptide SEQ ID NO. 6 led to an increase in the amount of insoluble tauin immunized rats compared to control rats, which received adjuvantalone. Immunoblot analysis revealed a trend towards an overall increasein insoluble phospho-tau levels (FIG. 43 and FIG. 26B). Immunization ledto an increase in total tau levels in the insoluble tau fraction, asrevealed by pan-tau mAb DC25. An increase was also observed at tauepitopes pT217 (33% increase), Thr231 (44% increase), and Thr181 (7%increase). However, AD-relevant epitope pS202/pT205 was an exception(FIG. 26B). The level of tau protein carrying this phosphoepitope wasreduced by 19% compared to control rats. Increased levels of multipledisease-relevant pathological insoluble tau protein indicate anundesirable negative effect of this vaccine on the rats.

The neurobehavioral response of rats treated with tau peptide SEQ IDNO:6/pS396/pS404 and controls was evaluated (FIGS. 44A, B, and C).Immunotherapy showed no statistically significant differences betweenthe treated and control rats in beam walking escape latency (p=0.82) orin the number of hind-limb slips (p=0.75). These results were confirmedby the Neuroscale score (p=0.96), in which no statistically significantdifferences in overall neurobehavioral performance were observed betweenimmunized and control rats. Thus, the treatment with peptide SEQ ID NO:6/pS396/pS404 had no statistically significant influence on the beamwalking test escape latency, number or hind-limb slips, or overall motorperformance of the tested rats.

FIG. 45 shows the percentage of transgenic rats with extensive taupathology, as assessed by immunohistochemistry with AT8. Immunotherapywith tau peptide SEQ ID NO:6/pS396/pS404 increased the tangle load by 9%relative to the control group. Thus, the negative effect of the SEQ IDNO:6/pS396/pS404 at the immunohistochemical level was confirmed at thebiochemical and neurobehavioral levels.

These results demonstrate that vaccination with tau phosphopeptide SEQID NO:6/pS396/pS404, which lacks any of the therapeutic tau epitopesdepicted in SEQ ID NO: 98-101, shows: 1) no therapeutic effect oninsoluble tau in the brains of immunized rats; 2) no effect at thebehavioral level; and 3) no reduction, but rather a 9% increase intangle load. Therefore, this phosphoepitope, which has been reported tobe AD-specific (Greenberg et al. 1992; Otvos et al. 1994), did notinduce a therapeutic effect.

h. SEQ ID NO:7 tau 181-TPPSSGEPPKSGDRSGYSSPGSPGTPGSRS-210 Carrying aPhosphorylated Serine Residue at Position 202 and a PhosphorylatedThreonine Residue at Position 205.

The resulting tau peptide SEQ ID NO:7/pS202/pT205, lacks any of thetherapeutic epitopes presented in the SEQ ID NO:98-101. Thephosphopeptide was conjugated to KLH and administered to transgenic rats(SHR72 line) in Freund's adjuvant

The quantitative analysis of tau immunoreactivity in the brain stem byimmunoblot did not show any therapeutic effect. (FIG. 46). The levels oftotal tau (DC25 epitope, 6% increase) and tau proteins phosphorylated ondisease-relevant tau epitopes pT217 (3% increase), Thr231 (11%increase), and Thr181 (7% increase), was slightly increased in immunizedrats compared to controls (FIGS. 46 and 26B). Immunization induced ahigher increase in insoluble tau protein phosphorylated at Ser202/Thr205(41% increase).

Immunotherapy with phosphorylated peptide SEQ ID NO: 7 did notsignificantly influence the sensorimotor function of the rats (FIG. 47).Immunized rats showed an apparent improvement in neurobehavioralparameters compared to controls. However, the groups were notstatistically different, as no statistically significant difference wasobserved between treated rats and controls in the beam walking escapelatencies (p=0.47, FIG. 47A), as well as in the number of hind-limbslips (p=0.54, FIG. 47B) and NeuroScale score (p=0.3, FIG. 47C).

Peptide SEQ ID NO:7 carries a phoshorylated tau epitope which does notcontain any of the DC8E8 epitopes. Examination of the brain stems of thetreated SHR72 rats revealed that the immunotherapy with tau peptide SEQID NO:7 was not able to reduce the neurofibrillary tangle load (FIG.48). Brain tissue from vaccinated and control animals contained nearlyidentical numbers of AT8 and DC217-positive NFTs in all areas of thebrain stem, mainly in the reticular formation. Vaccine containing SEQ IDNO:7, lacking DC8E8 epitopes, did not show any beneficial effect in thetreated animals.

These results demonstrate, that vaccination with tau phosphopeptide SEQID NO:7/pS202/pT205 produces: 1) no change in the levels of insolubletau in the brains of immunized rats at the biochemical level; and 2) noeffect at the behavioral level. This phosphopeptide provides additionalevidence that the phospho-sites represented by pS202/pThr205 are notsufficient to elicit an immune reaction eliminating pathological tauproteins and/or positively influencing the neurobehavioral status of therats. In contrast, the therapeutic tau epitopes (SEQ ID NOs:98-101)achieve this effect.

i. SEQ ID NO:8 tau 300-VPGGGSVQIVYKPVDLSK-317.

Immunotherapy with a shorter tau peptide, which was used as a controlbecause it does not carry a complete tau 6-mer within which one of thefour tau “therapeutic epitopes” could reside, nor a phosphorylatedepitope, was carried out to determine its ability to affect the levelsof pathological insoluble tau and neurofibrillary deposits in the brain.The level of total insoluble tau detected by pan-tau mAb DC25 wassignificantly increased (82%) in immunized rats compared to the controlsthat received adjuvant alone (FIG. 49 and FIG. 26B). Similarly,immunotherapy induced an increase in the levels of insoluble tauphosphorylated at T231 (60%) and a smaller increase in insoluble tauphosphorylated at position of T181 (10%). Quantitative analysis with adifferent set of antibodies, DC217 (pT217) and AT8 (pS202/pT205) did notreveal any effect on these epitopes in insoluble tau (FIG. 26B).

Animals were subjected to behavioral analysis (FIGS. 50A, B, C). Ratstreated with peptide SEQ ID NO: 8 showed no statistically significantdifference in escape latencies in the beam walking test (p=0.6) comparedto controls. The difference in the number of hind-limb slips was alsonot statistically significant (p=0.49) compared to controls. Similarly,there was no statistically significant difference in the NeuroScalescore (p=0.9). Thus, administration of the peptide SEQ ID NO: 8 did notstatistically significantly influence the motor performance of the rats.

These results demonstrate that vaccination with tau peptide SEQ ID NO:8,which lacks all of the therapeutic epitopes (within SEQ ID NOs:98-101),produced: 1) no change on insoluble tau in the brains of immunized ratsat the biochemical level; and 2) no effect at the neurobehavioral level.

The effect of the peptide vaccine on NFT load in brain stem of treatedand mock-control animals (received adjuvant only) was evaluated byimunohistochemistry. The results showed that immunotherapy with taupeptide SEQ ID NO:8 did not reduce the amount of neurofibrillary tangles(FIG. 51). Treated and mock-control animals developed nearly identicalnumber of neurodegenerative changes in all areas of the brain stem,mainly in the reticular formation of the brain stem as revealed by AT8and DC217 staining.

Vaccination with SEQ ID NO:8, which does not encompass any completetherapeutic epitopes (SEQ ID NO:98-101), did not show any beneficialeffect in the treated animals. Thus, the results of the immunizationswith peptides SEQ ID NO:5-8 show that the presence of at least onecomplete therapeutic epitope (located within SEQ ID NO:98-101) is neededfor the desired positive effect of the vaccine-reduction of taupathology and improvement in at least one neurobehavioral parameters.

j. SEQ ID NO:108 tau 294-KDNIKHVPGGGS-305. SHR72 rats were immunizedwith tau peptide SEQ ID NO:108 conjugated to the carrier KLH, formulatedwith alum adjuvant and administered in dose of 100 μg per animal.

Vaccination of transgenic rats SHR72 with tau peptide SEQ ID NO:108statistically significantly reduced insoluble pathological tau(p<0.001). Since the Alzheimer′ disease pathology in SHR 72 is caused bypathological insoluble tau forms (represented by monomers, dimers,oligomers and polymers of pathological tau), the impact of treatmentwith the 12-mer passive vaccine SEQ ID NO:108 on the insoluble taulevels was analyzed. The brain stem of the transgenic animals immunizedwith the respective immunogen and control group immunized with adjuvantalone were used for the extraction of sarkosyl-insoluble pathologicaltau (as described above). The results of the quantitative immunoblotanalysis from the group of transgenic rats immunized with tau peptideSEQ ID NO:108 and control group are shown in FIGS. 52 and 26B. Thevaccination statistically significantly reduced the pathologicalinsoluble tau in immunized animals compared to the control transgenicrats that received adjuvant alone (FIG. 52). This reduction was observedat all analyzed AD relevant tau epitopes. Immunization with tau peptideSEQ ID NO:108 induced significant reduction of insoluble pathologicaltau (p<0.001; 70%) revealed by measurement with pan-tau monoclonalantibody DC25 (FIG. 52). Moreover, analysis with phospho-dependent mAbsDC217 (pThr217) and AT8 (pSer202/pThr205) revealed significant reductionof the levels of pathological tau species phosphorylated at Thr217(p<0.001; 96%) and at Ser202/pThr205 (p<0.05; 98%) in insoluble tau ofimmunized rats compared to the controls (FIG. 52). Statisticallysignificant reduction was also observed at the levels of insolublepathological tau carrying pThr231 (p<0.05; 97%) and pThr181 (p<0.05;94%). These results show that the vaccine induced immune responseresults in statistically significant reduction of early forms ofpathological tau (represented by monomers, dimers, oligomers) and lateforms of pathological tau polymers (represented by PHFs).

Vaccination of Transgenic Rats SHR72 with Tau Peptide SEQ ID NO:108Statistically Significantly Improved Neurobehavioral Parameters(p<0.05).

Motor impairment was measured by the set of standard motor testscombined with the neurological examination in the compositescore—Neuroscale. At 6.5 months of age, transgenic rats SHR72 treatedwith tau peptide SEQ ID NO:108 were subject to behavioral tests with aimto determine the effect of immunotherapy. Rats treated with the taupeptide immunogen SEQ ID NO: 108 showed significantly decreased escapelatencies in the beam walking test (*p=0.04) compared to the transgenicrats that received adjuvant alone (controls) (FIG. 53). Similarly, apositive trend in number of hind-limb slips was observed in thevaccinated group in comparison with transgenic treatment controls, thisdifference was significant (*p=0.045, FIG. 53). The total Neuroscalescore was calculated from the data obtained in the beam walking tests,prehensile traction tests and neurological examinations (basic reflexes,hind-limb escape extension reflex). The immunization significantlyimproved the Neuroscale score of the rats treated with peptide SEQ IDNO: 108 compared to the control treatment group (*p=0.047) (FIG. 53C).The total Neuroscale score confirmed the neurobehavioral improvement oftreated transgenic rats when compared to untreated transgenic rats. Allstatistical data were obtained using nonparametric Mann-Whitney U-test.

Neurobehavioral parameters correlated with the insoluble pathologicaltau levels in the brain stem of the treated transgenic animals. Animalstreated with vaccine peptide SEQ ID NO: 108 exhibited low levels ofinsoluble pathological tau, which was associated with lower escapelatency and lower number of hind-limb slips in comparison with controltreatment animals. These findings show that the reduction of insolublepathological tau leads to the statistically significant improvements ofneurobehavioral deficits in the group of transgenic animals immunizedwith the 12-mer peptide (SEQ ID NO: 108) vaccine, showing itstherapeutic value.

Vaccination of Transgenic Rats SHR72 with Tau Peptide SEQ ID NO:108Resulted in 60% Reduction of Neurofibrillary Tangle (NFT) Load.

Immunohistochemical analysis of the neurofibrillary tau pathology(neurofibrillary tangles, NFT) in the brain stem showed reduction ofNFTs achieved in the vaccine treated SHR72 rats (FIG. 54). The number ofimmunized transgenic rats with extensive NFTs in the brain stem comparedto the adjuvant treated animals was decreased by more than 60%. Theimmunization reduced tau pathology (pathological tau polymers, PHFs) inthe brain of the transgenic animals immunized with SEQ ID NO: 108peptide vaccine.

The results show that immunotherapy with tau peptide SEQ ID NO:108efficiently reduced the tau pathology in SHR72 rats. Vaccination led toa statistically significant reduction in the insoluble pathological taulevels in the brains of the immunized animals as well as in thereduction of neurofibrillary tangle load (PHFs). The reduction in theamount of pathological tau proteins resulted in a statisticallysignificant improvement of neurobehavioral parameters of the treatedtransgenic rats. Thus, administration of tau peptide SEQ ID NO: 108 hascapacity for the treatment of AD.

Example 21 Immunotherapy with AD Therapeutic Peptides is Immunogenic andInduces the Production of Disease-Tau-Specific Antibodies in TransgenicRats

a. After five doses of tau peptide vaccine, analysis of the peptide'simmunogenicity in the treated rats was carried out. Sera from immunizedrats were used for antibody titer determination. Sera from ratsimmunized with adjuvant alone were used as a control. The titers ofspecific anti-tau antibodies were determined by ELISA, as described inExample 19. Serial dilutions of each serum were tested against ADtauΔ(1-150; 392-441)/4R and recombinant full-length tau 2N4R, previouslycoated onto microtiter well plates. The assayed immunogens induced theproduction of specific anti-tau antibodies.

For example, anti-tau specific antibodies were generated afterimmunization with 275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304 (SEQ ID NO: 4).Antibodies induced by tau peptide SEQ ID NO: 4 exhibited approximately3-fold higher binding activity to misdisordered tauΔ(1-150; 392-441)/4Rthan to tau 2N4R (FIG. 55; 1:3,200 dilution). These results furthersuggest that this vaccine induced antibodies which possess therapeuticpotential to recognize and eliminate/neutralize pathological tauproteins in Alzheimer's disease.

Also, vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:108 induced formation of antibodies preferentially binding topathological tau protein, over physiological tau. After five doses ofvaccine, analysis of the immunogenicity was done in each of thevaccinated rats. Animals were bled two weeks after the last booster doseand collected sera were used for geometric mean antibody titer (GMT)determination. The titers of specific anti-tau antibodies weredetermined by ELISA, as described in Example 19. Serial dilutions (1:100to 1:51,200) of each serum were tested against pathologicaltauΔ(1-150;392-441)/4R and physiological tau 2N4R, used as a solidphase. Titers were defined as the reciprocal of the dilution of serumthat gives half of the maximum Optical Density (Absorbance). Tocalculate geometric mean antibody titers, titer readings smaller than100 were assigned as value 10. As shown in FIG. 56, the antibody titerto pathological tauΔ(1-150;392-441)/4R (GMT 12800) was three-fold higherthan that to full-length tau 2N4R (GMT 4200). Antibody titers were alsomeasured against the unconjugated peptide SEQ ID NO:108 using the samemethodology described in Example 19. FIG. 56 shows that the highestantibodies titers were generated against tau peptide SEQ ID NO:108 (GMT20800). No antibody responses were observed in the transgenic ratsimmunized with adjuvant only (GMT 10; data not shown). Vaccination withpeptide SEQ ID NO:108, carrying DC8E8 epitope No. 2 (within SEQ ID NO:99), induced antibodies preferentially recognizing pathological tauprotein, thus discriminating between pathological tauΔ(1-150;392-441/4R)and physiological tau 2N4R. Moreover, results showed that SEQ ID NO:108is immunogenic and therefore possesses therapeutic potential toeliminate pathological tau proteins in Alzheimer's disease.

Moreover, vaccination of transgenic rats SHR72 with tau peptide SEQ IDNO:108 preferentially induced formation of IgG antibody isotypesspecific to pathological tau. To determine the specific isotypes of theantibodies produced in response to peptide SEQ ID NO:108, sera from ratsof the immunized and control groups were serially diluted from 1:100 to1:12,800, and tested in duplicates by ELISA (as described in Example 19)against pathological tauΔ(1-150; 392-441)/4R. To detect rat IgG1, IgG2a,IgG2b, IgG2c, and IgM isotypes, anti-rat subclass specificHRP-conjugated secondary antibodies were diluted 1:5,000 in PBS (Pierce,anti IgG1—PA1-84708, anti IgG2a—PA1-84709, anti IgG2b—PA1-84710,anti-IgG2—cPA1-84711 and anti-IgM—PA1-84712). FIG. 57 shows results forthe representative 1:800 dilution. The data demonstrate that the peptideSEQ ID NO:108 conjugated to KLH induced a broad spectrum of anti-tauantibody isotypes. The vaccine generated high levels of antibodyisotypes (IgG1, IgG2a, IgG2b and IgG2c), which are considered to be thehigh affinity antibodies. By contrast, isotypic profile showed very lowlevels of IgM antibodies. These are considered to have low affinity tothe antigen. The presence of high titers of IgG antibodies withpreferential affinity to pathological tau indicated that the immuneresponse induced by the vaccine is directed against pathological tauspecies. The control sera obtained from mock-immunized rats (whichreceived adjuvant alone) were negative.

These data were further used for determination of polarization (Th1/Th2phenotype) of the immune response. The levels of IgG1 and IgG2a isotypesinduced by immunization indirectly suggest the contributions of Th1cytokines versus Th2 cytokines to immune response. In general,production of IgG1 antibodies is induced by Th2 cytokines and productionof IgG2a antibodies is induced by Th1 cytokines. Therefore, the ratio ofIgG1 and IgG2a isotypes was calculated by dividing OD values for IgG1 byOD values for IgG2a. These data suggest (the ratio=0.625), that immuneresponse is slightly shifted toward Th1 phenotype.

b. Real time monitoring of binding events using surface plasmonresonance enabled measurement of the kinetic rate of association(k_(ON)) and dissociation (k_(OFF)) of antibodies from the pooled seraof rats immunized with tau peptide275-VQIINKKLDLSNVQSKCGSKDNIKHVPGGG-304 (SEQ ID NO:4). The analysisshowed a distinction between the recognition of tauΔ(1-150;392-441)/4Rand physiological tau isoform 2N4R (FIG. 58). The antibodies induced byimmunization exhibited a preferential affinity fortauΔ(1-150;392-441)/4R, binding this tau protein with three-times higheraffinity for truncated AD tau compared to their affinity for thecorresponding full-length isoform 2N4R. This binding difference wasdetermined to be, in part, due to the approximately five-times greaterk_(ON) rate for tauΔ(1-150;392-441)/4R (data not shown).

c. To further determine the specificity of antibodies induced in ratsimmunized with therapeutic tau peptides, the antisera can be used forimmunohistochemical staining of frozen sections of hippocampus fromhuman AD brain. For example, antibodies induced after immunization withSEQ ID NO: 4 were evaluated by this assay. The hippocampus from human ADbrain was fixed with 4% paraformaldehyde 4° C. for 2 days, followed by atreatment with 25% sucrose for 72 hours to provide cryoprotection. Thematerial was then frozen in cold 2-methylbutane (−42° C.) for 30 secondsand sectioned on cryomicrotome. Coronal sections (40 μm) were cut in acryostat at −18° C. Free-floating sections were used forimmunohistochemical studies. Free floating tissue sections were treatedwith cold (+4° C.) 99% formic acid for 1 min. at room temperature (25°C.). Brain sections were incubated for 20 minutes at room temperature in0.01 M of PBS, pH 7.4, containing 0.3% Triton X-100 and 1% H₂O₂,followed by a 30-minute incubation in the blocking solution (0.01 M PBS,containing 0.3% Triton X-100, 1% horse serum), followed by overnightincubation with sera from transgenic rats immunized with vaccinecontaining peptide SEQ ID NO: 4 (diluted 1:1000) at 4° C. After washing,the sections were immunostained using the standard avidin biotinperoxidase method (ABC Elite, Vector Laboratories, Burlingame, Calif.).The reaction product was visualized using avidin-biotin and Vector VIPas a chromogen (Vector Laboratories). Sections were then examined withan Olympus BX 51 microscope. Immunohistochemical staining showed thatthe antibodies induced by immunization with peptide SEQ ID NO:4specifically recognized pathological tau structures, i.e.neurofibrillary lesions in the hippocampus of Alzheimer's disease brain(FIGS. 59A, B). Sera from control rats, which received adjuvant alone,was used as a negative control, and it did not recognize any neuronalpathology (data not shown).

Vaccination of Transgenic Rats SHR72 with Tau Peptide SEQ ID NO:108Induced Antibodies Recognizing Pathological Tau Proteins in the Sectionsfrom the Human Alzheimer's Disease Brain Tissues.

To further determine the specificity of antibodies induced in ratsimmunized with therapeutic tau peptide SEQ ID NO:108, their sera wereused for immunohistochemical staining of the entorhinal cortex usingfrozen sections of human AD brain (Braak stage VI). Free floating tissuesections were incubated with sera (diluted 1:1000) from immunizedtransgenic rats at 4° C. The individual sera from the animals vaccinatedwith SEQ ID NO: 108 were used separately, while sera from animalsvaccinated with adjuvant only were pooled. After immunostaining usingthe standard avidin-biotin peroxidase method (ABC Elite, VectorLaboratories, Burlingame, Calif.) the sections were examined withOlympus BX 51 microscope. Immunohistochemical staining showed that theantibodies induced by immunization with peptide SEQ ID NO:108specifically recognized pathological tau structures, i.e.neurofibrillary lesions in the entorhinal cortex of Alzheimer's diseasebrain. FIG. 60 (A-E) shows representative immunostaining with the ratsera collected from five vaccinated transgenic rats SHR72. Sera of theanimals vaccinated with SEQ ID NO:108 decorated the neurofibrillarypathology very intensively, confirming that the antibodies efficientlytargeted pathological tau proteins. Sera from control rats, whichreceived adjuvant alone, were used as negative controls. They did notrecognize any neurofibrillary pathology (FIG. 60F).

Antibodies Induced by the SEQ ID NO:108 Vaccine Recognize PathologicalTau Proteins Extracted from Brains of SHR72 and from Human AD Brains.

The specificity of the sera from rats immunized with tau peptide SEQ IDNO:108 was further examined on pathological forms of soluble andinsoluble pathological tau using immunoblot method (as described inExample 19). The brain stems of SHR72 rats in the late stage of thepathology were used for the extraction of soluble insoluble pathologicaltau proteins. Temporal cortex of human AD brain (Braak stage VI;obtained from the Netherlands Brain Bank, Netherlands) was used for theextraction of human pathological AD tau. The soluble and insolublepathological tau proteins were prepared using the same method asdescribed in Example 8. For soluble tau fractions 15 μg of totalproteins were loaded per lane. For insoluble tau fractions the pelletswere dissolved in 1× sodium dodecyl sulfate (SDS) sample loading buffer(Laemmli, 1970) in 1/50 volume of the soluble fraction used for thepreparation of the insoluble tau fraction and equal volume was loadedonto SDS-PAGE. Pooled sera from immunized animals were diluted 1:1000 inPBS and used as a primary antibody. Incubation with primary antibody wasfollowed by polyclonal rabbit anti-rat immunoglobulins conjugated tohorseradish peroxidase (1:3000; Dako, Glostrup, Denmark). Western blotsignal was digitized with LAS3000 CCD imaging system (Fujifilm, Japan).The results of this immunoblot analysis are shown in FIG. 61.

The results (FIG. 61) show that antibodies generated against peptidecarrying DC8E8 epitope 2 (SEQ ID NO:99) recognize the pathological tauproteins extracted from SHR72 and from AD brains tissues. The inducedantibodies recognized monomeric forms of pathological tau (lane No. 1, 2and No. 3) as well as oligomeric forms of pathological tau (lane No. 2and No. 3) including the A68 tau triplet characteristic for AD. Thesefindings have key impact on immunotherapy using this rat model ofAlzheimer's disease. High affinity antibodies generated by vaccinetarget all forms of pathological tau proteins. They target monomericforms of pathological tau proteins and thus prevent pathological tau-tauinteraction (oligomerization) leading to reduction of insoluble taulevels in vaccinated rat and consequently to improvement ofneurobehavioral parameters. The generated antibodies also bindoligomeric forms of pathological tau and target them for degradation,e.g. by microglia, as it was described in Example 10 for mAb DC8E8.

Example 22 Immunotherapy with Ad Therapeutic Peptides Induces theProduction of Disease-Tau-Specific Antibodies in Mice

Peptides SEQ ID NO:109, SEQ ID NO:110 (SEQ ID NO: 88 corresponds to SEQID NO: 110 μlus an additional N-terminal Cys for conjugation), SEQ IDNO:111, and SEQ ID NO:112, carrying one of the therapeutic epitopeswithin either SEQ ID NO: 100 or 101, induced production of antibodies inimmunized mice. The resulting antibodies show statisticallysignificantly higher binding activity to pathological tauΔ(1-150;392-441)/4R than to physiological tau 2N4R.

SEQ ID NO: 109 Tau 314-DLSKVTSKCGSLGNIHHKPGGGQVEVKSE-342 SEQ ID NO: 110Tau 352-SKIGSLDNITHVPGGGNKKIETHKLTFREN-381 SEQ ID NO: 111Tau 325-LGNIHHKPGGGQ-336 SEQ ID NO: 112 Tau 357-LDNITHVPGGGN-368SEQ ID NO: 100 Tau 329-HHKPGGG-335 SEQ ID NO: 101 Tau 361-THVPGGG-367

Indeed, with the aim to further determine the immunogenic potential ofpeptides carrying one or more therapeutic DC8E8 epitopes (e.g., within7-mer: SEQ ID NO: 100 and SEQ ID NO: 101), twelve and thirty amino acidslong peptides (SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 and SEQ IDNO:112) carrying one of the DC8E8 therapeutic epitopes were designed.Tau peptides SEQ ID NO:109 (30 amino acids) and SEQ ID NO:111 (12 aminoacids) contain within them the therapeutic epitope in SEQ ID NO:100. Taupeptides SEQ ID NO:110 (30 amino acids) and SEQ ID NO:112 (12 aminoacids) contain within them the therapeutic epitope in SEQ ID NO:101.Peptides were conjugated to KLH via their N-terminal Cys residue asdescribed in Examples 18-19. Vaccines for immunizations were preparedwith peptide-KLH conjugates, containing 100 μg of conjugated peptide, in100 μl of PBS and emulsified 1:1 (vol/vol) with Freund's adjuvant in afinal dose volume of 200 μl. Five Balb/c mice were used per treatmentgroup. The first immunization was done using the peptide-conjugate inPBS formulated with Freund's complete adjuvant. The two followingimmunizations, in two-week intervals, were performed using thepeptide-conjugate in PBS formulated with Freund's incomplete adjuvant.As a control, vaccine containing adjuvant-only was used. Sera werecollected 10 days after the last immunization and antibody response weremeasured by ELISA as described in Example 19. Sera from individual micewere serially diluted from 1:100 to 1:12,800 and tested in duplicates.To determine the specificity of sera, the pathologicaltauΔ(1-150;392-44114R) and physiological tau 2N4R were used as the solidphase. FIGS. 62 through 65 show a summary of the results for sera at1:800 dilution. The ELISA results were statistically evaluated using theMann-Whitney non-parametric test.

All tested peptides generated tau-specific antibodies in immunized mice.Antibodies induced by vaccinations with tau peptides SEQ ID NO:109, SEQID NO:110, SEQ ID NO:111, and SEQ ID NO:112 exhibited statisticallysignificantly higher binding activity to pathological tauΔ(1-150;392-441)/4R than to physiological tau 2N4R (FIG. 62-65). Moreover,shortening of the peptides from 30 amino acids (SEQ ID NO:109, SEQ IDNO:110) to 12 amino acids (SEQ ID NO:111 and SEQ ID NO:112) led tosignificantly higher production of specific antibodies, whichpreferentially recognized pathological tau (SEQ ID NO:109, p=0.0115; SEQID NO:110, p=0.0029; SEQ ID NO:111, p=0.0007; SEQ ID NO:112, p<0.001).Altogether, these results showed that the peptides SEQ ID NO:109 and SEQID NO:111 (carrying the therapeutic epitope within SEQ ID NO:100) andpeptides SEQ ID NO:110 and SEQ ID NO:112 (carrying the therapeuticepitope within SEQ ID NO:101) are immunogenic and possess therapeuticactivity targeting pathological tau proteins present in the brains ofpatients suffering from Alzheimer's disease.

Example 23 Identification of Designer Peptides (Designer TherapeuticEpitopes) Capable of Competing with Pathological Tau for Binding to atLeast One Dc8E8 Epitope

Two additional peptides (11-mers) were designed based on the conservedamino acid residues between epitope #1 (within KHQPGGG, SEQ ID NO:98),#2 (within KHVPGGG, SEQ ID NO:99), #3 (within HHKPGGG, SEQ ID NO:100)and #4 (within THVPGGG, SEQ ID NO:101). In their design, five residuescontributing for the binding of DC8E8 to these epitopes were kept fixed:histidine, proline, and three glycine residues in the sequence HxPGGG(SEQ ID NO: 164). Peptides were synthesized by EZBiolabs (USA) withpurity higher than 85%. The two designer therapeutic epitopes areGWSIHSPGGGSC (SEQ ID NO: 250) and SVFQHLPGGGSC (SEQ ID NO: 251).

These peptides were analyzed for their ability to compete withpathological tau by competition ELISA. ELISA plates (IWAKI high bindplate, #3801-096, Bertoni GmbH, Austria) were coated with 100 μl/well of5 μg/ml of recombinant purified tauΔ(1-150;392-441)4R in PBS overnightat 4° C. The coated ELISA plates were washed 4 times with PBS/Tween 20(phosphate-buffered saline supplemented with 0.05% v/v Tween 20), andblocked with PBS/Tween 20 for 2 h at 25° C. Each of the peptides wasseparately dissolved in PBS at the final concentration of 5 mM. Serial2-fold dilutions of the peptides in PBS/Tween 20 in polypropylene plates(Greiner, #651201) were prepared (concentration range 80 μM, 40 μM, 20μM, 10 μM, 5 μM, and 2.5 μM). 100 μl of each peptide dilution was mixedwith 100 μl of 2 μg/ml purified DC8E8 monoclonal antibody (purificationwas done as described in Example 5). The resulting 200 μl of mixturethen contained 1 μg/ml DC8E8 antibody and 40 μM, 20 μM, 10 μM, 5 μM, 2.5μM and 1.25 μM peptides. tauΔ(1-150;392-441)4R was included as apositive control. The antibody/peptide mixtures were incubated for 1 hrat 25° C. on a rotating platform set to 250 rpm. One hundred microlitersof the antibody/peptide mixtures were then transferred into the preparedELISA plates and incubated for 1 hr at 25° C. on a rotating platform setto 250 rpm. The ELISA plates were washed 4× times with PBS/Tween 20. TheELISA plates were then incubated with 100 μl of Polyclonal GoatAnti-Mouse Immunoglobulins/HRP (Dako, #P0447) diluted 1:4 000 inPBS/Tween 20 and incubated for 1 hr at 25° C. on a rotating platform setto 250 rpm. The ELISA plates were washed 4× times with PBS/Tween20. TheELISA plates were then incubated with 100 μl of 1.5 mg/2 ml o-PDA(o-phenylenediamine, SIGMA, P1526) in 0.1 M Na-Acetate pH=6.0 (Roth,#6779) supplemented with 1.5 μl/2 ml of 30% H₂O₂ (SIGMA, H-0904) for 10minutes at 25° C. in dark. The reaction was stopped by adding 100 μl of2M H₂SO₄ (Merck, 1.00731.1000). The developed signal was measured byreading at 490 nm (e.g. using the Victor Multilabel Counter (Wallac).

The two designer peptides, namely GWSIHSPGGGSC (SEQ ID NO: 250) andSVFQHLPGGGSC (SEQ ID NO: 251), were both able to compete withtauΔ(1-150;392-44)4R for binding to DC8E8 (FIG. 66). The peptideGWSIHSPGGGSC (SEQ ID NO: 250), termed designer therapeutic epitope 1,showed the most similar affinity for DC8E8 compared totauΔ(1-150;392-441)4R. The affinity of the peptide SVFQHLPGGGSC (SEQ IDNO: 251), termed designer therapeutic epitope 2, for DC8E8 was more thanone order of magnitude higher than the affinity of disease tau(tauΔ(1-150;392-441)4R for this antibody.

Example 24 In Vivo Testing of Designer Therapeutic Epitopes 1 and 2 forImmunogenicity and Specificity of the Immune Response

The designer therapeutic epitope 1 (GWSIHSPGGGSC, SEQ ID NO: 250) anddesigner therapeutic epitope 2 (SVFQHLPGGGSC, SEQ ID NO: 251) wereconjugated to KLH via their C-terminal Cys residue (as described inExample 18) and used to immunize Balb/c mice. Three mice were used foreach designer therapeutic epitope. Immunizations were done as follows.Vaccines for immunizations were prepared with designer therapeuticepitope-KLH conjugates, containing 100 μg of the conjugated peptide in100 μl of PBS and emulsified 1:1 (vol/vol) with Freund's adjuvant in afinal dose volume of 200 μl. The first immunization was done using thedesigner therapeutic epitope-KLH conjugate in PBS formulated withFreund's complete adjuvant. The four following immunizations, infour-week intervals, were performed using the designer therapeuticepitope-KLH conjugates in PBS formulated with Freund's incompleteadjuvant. For control immunization, PBS was used instead of designertherapeutic epitope-conjugates. Sera were prepared 14 days after thelast immunization.

1. Antibodies Discriminate Pathological Tau.

To determine the specificity of sera, two tau proteins were used:recombinant pathological tauΔ(1-150;392-441)4R and physiological Tau2N4R. The sera from each mouse were serially diluted from 1:100 to1:12,800 and tested in triplicates. Antibody titers were determinedusing Polyclonal Goat Anti-Mouse Immunoglobulins/HRP (Dako, #P0447)diluted 1:4,000. FIG. 67 shows representative results for 1:3200dilution.

Both designer therapeutic epitopes generated a high immune response inimmunized mice. Furthermore, both tested designer therapeutic epitopesinduced antibodies that recognized pathological tauΔ(1-150;392-441)4Rwith higher affinity compared to physiological Tau 2N4R (FIG. 67). Thisdiscrimination is statistically significant for sera from all immunizedanimals. Altogether, and in combination with the results obtained withthe activity observed with DC8E8, these results show that both designertherapeutic epitopes 1 and 2 are immunogenic and induce antibodyresponse with therapeutic potential to target pathological tau proteinsin the brains of Alzheimer's disease patients.

2. Antibody Isotype:

To determine the specific isotypes of the antibodies produced inresponse to these designer therapeutic epitopes 1 and 2, sera from miceof the same group were pooled, serially diluted from 1:100 to 1:12,800,and tested in triplicates by antibody isotype ELISA. To detect mouseIgM, IgG1, IgG2a, IgG2b, and IgG3 isotypes, anti-mouse subclass specificHRP-conjugated secondary antibodies were used (antibodies purchased fromLifespan Biosciences, anti IgG1—#LS-059107, anti IgG2a—# LS-059112, antiIgG2b—#LS-C59117, anti-IgG3—#LS-059125 and anti-IgM—#LS-055875).Antisera obtained from control mice were negative. FIG. 68 showsrepresentative results for 1:800 dilution. The data obtained with pooledsera demonstrated that immunization with both tested designertherapeutic epitopes induced a broad spectrum of anti-tau antibodyisotypes and that the isotype profile was very similar in all tests.Both designer therapeutic epitopes generated mainly IgG1 antibodies incomparison with IgG2a, IgG2b and IgG3 responses. (FIG. 68).

3. Designer Therapeutic Epitopes Induce Antibody Response StatisticallyHighly Significantly Discriminating Between Pathological andPhysiological Tau.

Analysis of the affinities of the antibodies generated against designertherapeutic epitopes was done by surface plasmon resonance onBIACORE3000 using a CM5 sensor chip (Biacore AB, Uppsala) as describedin Examples 5 and 19. In each analysis cycle, mouse antiserum againsteither GWSIHSPGGGSC (SEQ ID NO: 250), designer therapeutic epitope 1(diluted 100-fold) or SVFQHLPGGGSC (SEQ ID NO:251), designer therapeuticepitope 2 (diluted 100-fold), (pooled antisera from 3 mice), wascaptured in the analytical flow cell to reach immobilization level ˜950RU, which approached saturation. As a reference, an irrelevant antibodyRab50 (Macikova et al., 1992), which does not bind tau, was captured inthe reference flow cell. For the K_(A) determination as well as for thedetermination of the kinetic rate constants k_(ON) and k_(OFF), 100 nMsolutions of either pathological tauΔ(1-150;392-441)4R or physiologicalTau 2N4R, were injected at a flow rate 100 μl/min over the sensor chip.

The antibodies induced by vaccination with designer therapeutic epitopes1 and 2 discriminated between pathological tauΔ(1-150;392-441)4R andphysiological Tau 2N4R (FIGS. 69A and 69B). Affinity of antibodiespresent in antiserum against designer therapeutic epitope 1(GWSIHSPGGGSC, SEQ ID NO: 250) measured by surface plasmon resonance,exhibited nearly 50-times higher affinity to pathological tau, comparedto physiological tau, which is highly statistically significant(p<0.001). Affinity of antibodies present in antiserum against designertherapeutic epitope 2 (SVFQHLPGGGSC (SEQ ID NO:251) measured by surfaceplasmon resonance, exhibited nearly 15-times higher affinity topathological tau, compared to physiological tau, which is highlystatistically significant (p<0.01).

4. Designer Therapeutic Epitopes Induce Antibody Response RecognizingPathological Tau Species in Human AD Brains.

To determine the specificity of the antibodies generated in miceimmunized with designer therapeutic epitopes, immunohistochemicalstaining was done on frozen sections of human AD brains.

The human AD brain tissue samples (entorhinal cortex, AD Braak VI,provided by the Netherlands Brain Bank) were fixed with 4%paraformaldehyde in PBS for 2 days at 4° C. and then cryoprotected (25%sucrose), frozen in cold 2-methylbutane (−42° C.) and sectioned oncryotome. Free floating tissue sections (40 μm) were treated with cold(4° C.) 99% formic acid for 1 min at room temperature (25° C.). Thesections were immunostained using the standard avidin-biotin peroxidasemethod (ABC Elite, Vector Laboratories, Burlingame, Calif.). Mouseantisera against designer therapeutic epitope 1 (SEQ ID: 250) anddesigner therapeutic epitope 2 (SEQ ID NO: 251), each pooled from 3immunized mice, were diluted 1:2000 in the blocking solution (5% bovineserum albumin, 0.3% Triton X-100 in PBS). Sections were then examinedwith an Olympus BX 51 microscope.

Immunohistochemical staining showed that mouse immunosera generatedagainst both designer therapeutic epitopes GWSIHSPGGGSC (SEQ ID NO: 250)and SVFQHLPGGGSC (SEQ ID NO: 251) specifically recognized pathologicaltau structures, i.e. neurofibrillary tangles and neuropil threads, inthe entorhinal cortex of Alzheimer's disease brain (FIG. 70A-D).Antisera against designer therapeutic epitope 1 and designer therapeuticepitope 2 did not recognize normal tau in the control human brain. (FIG.70E, F).

5. Designer Therapeutic Epitope 2 (SVFQHLPGGGSC, SEQ ID NO: 251) InducesAntibody Response Recognizing Pathological Tau Species in the Brains ofTransgenic Rat Model of Alzheimer's Disease.

To determine the specificity of the antibodies generated in miceimmunized with designer therapeutic epitopes, immunohistochemicalstaining was done on paraffin embedded sections of the brains oftransgenic rats SHR72.

Transgenic rats of the strain SHR72 (7 months old) were perfusedtranscardially with PBS for 1 min under deep anesthesia followed byperfusion with 100 ml of 4% paraformaldehyde (pH 7.4). After perfusion,the head was cut off and the brain was quickly removed. The brain wascut sagittally into two equal-sized hemispheres using disposable scalpelblades. The brain tissues were post-fixed in 4% paraformaldehyde,embedded in paraffin, and cut into sections on a microtome.Immunohistochemistry and histopathology were done on 8 μmparaffin-embedded tissue sections. Tissue sections were pre-treated for20 min with an antigen unmasking solution (Vector laboratories, CA, USA)and for 1 min with cold (+4° C.) 90% formic acid (Applichem, Germany),at room temperature (25° C.). After blocking, the sections wereincubated overnight with serum generated against designer therapeuticepitope 2 (SVFQHLPGGGSC, SEQ ID NO: 251) that was diluted 1:1000 inblocking solution (5% bovine serum albumin, 0.3% Triton X 100 in 50 nMTris-HCl). After washing, the sections were incubated with abiotinylated secondary antibody (Vectastain Elite ABC Kit, VectorLaboratories) at room temperature for an hour, and then reacted with anavidin-biotin peroxidase-complex solution for 60 minutes (VectastainElite ABC Kit, Vector Laboratories), at room temperature (25° C.). Theimmunoreaction was visualized with a peroxidase substrate kit (VectorVIP, Vector laboratories, Ca, USA). Sections were examined with anOlympus BX71 microscope.

In the transgenic rat brain (SHR72), serum generated against designertherapeutic epitope 2 (SVFQHLPGGGSC, SEQ ID NO: 251) recognizedneurofibrillary tangles (FIG. 70G). In age-matched control rat brainsthe antibody did not stain neuronal cells (FIG. 70H).

Serum generated against designer therapeutic epitope 2 (SVFQHLPGGGSC,SEQ ID NO: 251) recognized oligomeric pre tangle stage tau (FIG. 70I),as well as intracellular neurofibrillary tangles (FIG. 70J).

6. Antibodies Induced by Designer Therapeutic Epitopes 1 and 2 RecognizeSoluble and Insoluble Pathological Tau in Human AD Brain:

Sarkosyl soluble and insoluble pathological tau was isolated from thetemporal cortex of human Alzheimer's disease (obtained from theNetherlands Brain Bank) and analyzed by immunoblotting, as described inExample 8.

The membrane containing soluble and insoluble pathological tau proteinfractions were incubated either with DC8E8 hybridoma supernate diluted1:1 with 5% non-fat dry milk in PBST or with pooled mouse antiseragenerated against designer therapeutic epitope 1 (SEQ ID NO: 250,GWSIHSPGGGSC) or with pooled mouse antisera generated against designertherapeutic epitope 2 (SVFQHLPGGGSC, SEQ ID NO: 251) whereby both pooledantisera were diluted 1:100 in 5% non-fat dry milk in PBST. Membraneswere washed and then incubated with peroxidase-conjugated goatanti-mouse IgG (DAKO, Denmark) diluted 1:4000. The blots were developedwith SuperSignal West Pico Chemiluminescent Substrate (Pierce, U.S.A)detected using the LAS3000 imaging system (FUJI Photo Film Co., Japan).The signal intensities were quantified using AIDA software (AdvancedImage Data Analyzer, Raytest, Straubenhardt, Germany).

The results of this immunoblot analysis are shown in FIG. 71. Theseresults show that antibodies generated against both designer therapeuticepitopes 1 (i.e., GWSIHSPGGGSC) (SEQ ID NO: 250) and 2 (i.e.SVFQHLPGGGSC) (SEQ ID NO: 251) recognize the same pathological tauproteins as DC8E8. All the GWSIHSPGGGSC (SEQ ID NO: 250) antisera,SVFQHLPGGGSC (SEQ ID NO: 251) antisera and DC8E8 antibody specificallyrecognized pathological tau proteins present in sarkosyl soluble andinsoluble tau fraction isolated from AD brain tissues (FIG. 71).

7. Antibodies Induced by Designer Therapeutic Epitopes 1 and 2 RecognizeSoluble and Insoluble Pathological Tau in Brains of Tau Transgenic Rats.

Sarkosyl soluble and insoluble pathological tau was isolated from thebrains of tau transgenic rat brains (SHR72 line described in Example 7)as described in Example 8.

The results of the immunoblot analysis (described in the Example 8) areshown in FIG. 72. These results show that antibodies induced by designertherapeutic epitopes 1 (i.e., GWSIHSPGGGSC) (SEQ ID NO: 250) and 2 (i.e.SVFQHLPGGGSC) (SEQ ID NO: 251) recognize the same pathological tauproteins as DC8E8 (FIG. 72). Targeting of monomeric and oligomericpathological tau protein prevent generation of pathological tauaggregates resulting in decreased tau pathology leading to therapeuticeffect and treatment of AD in human.

Example 25 In Vivo Efficacy of Designer Therapeutic Epitopes inTransgenic Rats Modeling Ad

Immunotherapy with Designer Therapeutic Epitopes Showed Improvement inNeurobehavioral Parameters of Treated Rats.

Designer therapeutic epitope 2 (SEQ ID NO: 251) was selected for theimmunotherapy in transgenic rats SHR 72. Rats were immunizedsubcutaneously with vaccine doses containing designer therapeuticepitope 2 (SEQ ID NO: 251) conjugated to KLH combined with Adju-phosadjuvant. Vaccines were prepared as described in Example 18. One dosecontained 100 μg of conjugated designer therapeutic epitope 2. Complexmotor impairment was measured by the set of standard motor testscombined with the neurological examination in the compositescore—Neuroscale. At 6.5 months of age, transgenic rats SHR72 treatedwith vaccine containing designer therapeutic epitope 2 (SEQ ID NO: 251)were subjected to behavioral tests with aim to determine the effect ofimmunotherapy.

Rats treated with the designer therapeutic epitope 2 (SEQ ID NO: 251)showed decreased escape latencies by 27% in the beam walking test thanthe transgenic control rats that received adjuvant alone (FIG. 73A). Thenumber of the hind-limb slips was statistically significantly reduced(p<0.05) by 44% in the vaccinated group in comparison with transgeniccontrols (FIG. 73B). The Neuroscale score was calculated from the valuesobtained in the beam walking tests, prehensile traction tests andneurological examinations (basic reflexes, hind-limb escape extensionreflex). The immunization significantly improved the Neuroscale score ofthe rats treated with peptide SEQ ID NO: 251 by 26% compared to thecontrol group (FIG. 73C). Total Neuroscale score confirmed theneurobehavioral improvement of treated transgenic rats when compared tountreated transgenic rats. All statistical data were obtained usingnonparametric Mann-Whitney U-test.

Immunotherapy with Designer Therapeutic Epitope 2 Showed StatisticallySignificant Reduction (p<0.05) of Pathological Tau in the Brains ofTreated Alzheimer Transgenic Rats.

To confirm the effect of the immunization with designer therapeuticepitope 2 (SEQ ID NO: 251) on the levels of insoluble pathological tau,we used immunoblot analysis of the rat brain samples. Brain tissue (thebrain stem) of the transgenic animals immunized with designertherapeutic epitope 2 (SEQ ID NO: 251) and control group of transgenicanimals immunized with adjuvant alone were used for the preparation ofsarkosyl-insoluble tau fraction as described in Example 8. Immunoblotanalysis was done as described in Example 19. The statistical analysiswas done by T-test. Phosphorylation dependent monoclonal antibodies AT8,DC209, DC217 and pan-tau monoclonal antibody DC25 were used in thestudy.

The results of immunoblot quantitative analysis of insoluble tau levelsfrom the group of transgenic rats immunized with designer therapeuticepitope 2 (SEQ ID NO: 251) and control group are shown in FIG. 74.Immunotherapy reduced statistically significantly the amount ofinsoluble tau in immunized animals compared to the control transgenicrats that received adjuvant alone. The reduction of insoluble tau wasobserved at all analyzed tau epitopes. Reductions at 347-353 epitope andphospho-tau epitopes were statistically significant (P<0.05). Observedreduction was as follows: at 347-353 tau epitope by 46% (P<0.05), atpT217 tau epitope by 57% (P<0.05), at p231-tau epitope by 55% (P<0.05),at pS202/pT205 tau epitope by 47% (P<0.05).

These results show that the vaccine induced pathological tau specificantibodies which led to reduction of pathological tau. The reduction ofpathological tau levels in the brain of treated Alzheimer's disease ratmodel correlated with neurobehavioral parameters. Treated animals withlow levels of insoluble tau showed shorter escape latency andstatistically significantly reduced number of hind-limb slips (p<0.05),in comparison with control animals. These findings show that theimmunization with designer therapeutic epitope leads to the reduction ofinsoluble pathological tau and to neurobehavioral improvement in treatedanimals, which underlines the therapeutic potential of the vaccine forthe treatment of the human Alzheimer's disease and related tauopathies.

Example 26 Further Characterization of Dc8E8 Minimal Epitopes (forTherapeutic Core Units)

To further characterize DC8E8's minimal epitopes, a panel of taupeptides with different lengths (42-mer, 19-mer, 12-mers, 7-mers, 6-mersand 5-mers), derived from the microtubule binding repeat regions (MTBR1,MTBR2, MTBR3, MTBR4) of human tau protein 2N4R were designed (FIG. 75 A,B). Peptides were synthesized by EZBiolabs (USA) with purity higher than95%. All peptide were analyzed for their ability to compete withpathological tauΔ(1-150;392-441)/4R for binding to DC8E8 by competitionELISA. ELISA plates (Sarstedt, #821581001) were coated with 50 μl/wellof 5 μg/ml of recombinant purified tauΔ(1-150;392-441)/4R) in PBSovernight at 37° C. The coated plates were washed 5 times with PBS/Tween20 (0.05% v/v), and blocked with PBS/Tween 20 (0.05% v/v) for 1 h at 25°C. Each of the peptides was separately dissolved in PBS at a finalconcentration of 1 mM. Serial dilutions (2.5x) of peptides in PBS/Tween20 were prepared in polypropylene microtiter plates with conical wellbottom (Greiner, #651201) within the concentration range of 200 μM; 80μM; 32 μM; 12.8 μM; 5.12 μM; 2.048 μM; 0.8192 μM; 0.32768 μM). Thevalidation monoclonal antibody DC8E8 was diluted to a concentration of0.6 μg/ml in PBS and 60 μl of this diluted antibody was added into eachwell to serial dilution of peptides resulting in 120 μl/well of mixture.The antibody/peptide mixtures were incubated for 1 hr at 25° C. on arotating platform set to 230 rpm. 50 μl/well of antibody/peptidemixtures were transferred from polypropylene plates intotauΔ(1-150;392-441)/4R coated and PBS/Tween 20 blocked ELISA plates (induplicates) and incubated for 1 hr at 25° C. The plates were washed 5×times with PBS/Tween 20 and incubated with 50 μl/well of Polyclonal GoatAnti-Mouse Immunoglobulins/HRP (Dako, #P0447) diluted 1:1000 inPBS/Tween 20 for 1 hr at 25° C. After washing, the plates were thenincubated with 50 μl/well of 1 mg/2 ml o-PDA (o-phenylenediamine, Sigma,P1526) in 0.1 M Na-Acetate pH=6.0 (Roth, #6779) supplemented with 1.5μl/2 ml of 30% H₂O₂ (Sigma, H-0904) for 20 minutes at 25° C. in dark.The reaction was stopped by adding 50 μl/well of 2M H₂SO₄ (Merck,1.00731.1000) followed by reading the plates at 492 nm (e.g. PowerwaveHT, Bio-Tek).

FIG. 76 shows the results of the competition ELISA performed with thefollowing peptides: TENLKHQPGGGK (SEQ ID NO: 270), KHQPGGG (SEQ ID NO:271), HQPGGG (SEQ ID NO: 272), HQPGG (SEQ ID NO: 273), QPGGG (SEQ ID NO:274), ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS (SEQ ID NO: 275),KHVPGGG (SEQ ID NO: 276), HVPGGG (SEQ ID NO: 277), HVPGG (SEQ ID NO:278), VPGGG (SEQ ID NO: 279), DNIKHVPGGGSVQIVYKPV (SEQ ID NO: 280),HHKPGGG (SEQ ID NO: 281), HKPGGG (SEQ ID NO: 282) and THVPGGG (SEQ IDNO: 283). All analyzed peptides that encompassed tau therapeuticepitopes competed with pathological tauΔ(1-150;392-441/4R). As shown inFIG. 76, even 6-mer peptides (SEQ ID NO: 272, SEQ ID NO: 277 and SEQ IDNO: 282) were able to compete with tau Δ(1-150;392-441)/4R for bindingto DC8E8. However, removal of either histidine (5-mer peptides SEQ IDNOs: 274, 279) or the last glycine (5-mer peptides SEQ ID NOs: 273, 278)led the to a loss of competing activity with tauΔ(1-150;392-441/4R) forbinding to DC8E8 (the 5-mer peptides were derived from SEQ ID NO: 272and SEQ ID NO: 277 with removed amino acids His and Gly underlined, seeabove). These results suggest that the 5-mers peptides did not generatethe therapeutic 3D structure that is recognized by DC8E8 on pathologicaltau. On the other hand, peptides comprising six amino acid residuescreate therapeutic 3D structure responsible for the biological activitymeasured by competitive ELISA and form minimal epitopes (therapeuticcore units) of DC8E8. In their design, five amino acid residues areimportant for DC8E8 recognition, which are conserved (histidine,proline, and the three glycine residues in the sequence HxPGGG).

Conclusion:

The data suggest that the minimal DC8E8 epitope on human tau consists of6 amino acids, which comprise the residues HQPGGG (located withinMTBR1), HVPGGG (within MTBR2), HKPGGG (within MTBR3) and HVPGGG (withinMTBR4). Thus, the DC8E8 binding site (=epitope) is present four times in2N4R tau and three times in 2N3R tau. This suggests that the requiredamino acids within the 6-mer sequence are the histidine and all threeglycines.

Example 27 Determination of the Immunogenicity of Peptides CarryingDC8E8 Minimal Epitopes

a) Peptides Carrying DC8E8's Minimal Epitopes are Immunogenic:

With the aim to determine the immunogenic potential of individual taupeptides, peptides were conjugated to KLH via their N-terminal Cysresidue.

Towards this end, tau peptides were synthetized as cysteinated peptideswith an extra N-terminally located cysteine residue with the aim toobtain oriented attachment of the peptide on the surface of the KLHprotein. Peptides were coupled to the KLH carrier via bifunctionalcross-linker N-[γ-maleimidobutyryloxy]succinimide ester (GMBS). Toprepare the conjugation reaction, 20 mg of KLH (Calbiochem) weredissolved in conjugation buffer (PBS with 0.9 M NaCl, 10 mM EDTA) to aconcentration of 10 mg/ml by gentle mixing for 10 minutes. Forpreparation of maleimide-activated KLH, 2 mg of active bi-functionalcross-linker GMBS were dissolved in 50 μl of anhydrous dimethylformamideand mixed with 2 ml of KLH solution for 1 hour at room temperature.Subsequently, un-reacted GMBS was removed on a 5 ml HiTrap Desaltingcolumn (GE Healthcare) equilibrated in conjugation buffer. Conjugationswere carried out at a 1:1 ratio of peptide to maleimide-activated KLH(w/w, 20 mg of peptide) for 2 h at room temperature (25° C.). Theresulting conjugates were dialyzed against a 100-fold excess of PBS,with four dialysis buffer changes to remove unconjugated peptide. Afterdialysis, the conjugates were centrifuged at 21,000×g for 15 min at 2°C. The conjugates were aliquoted and stored at −20° C. until used.

Vaccines for immunizations were prepared with peptide-KLH conjugates,containing 100 μg of conjugated peptide, in 100 μl of PBS and emulsified1:1 (vol/vol) with Freund's adjuvant in a final dose volume of 200 μl.Five C57/BL mice were used per treatment group. The first immunizationwas performed using the peptide-conjugate in PBS formulated withFreund's complete adjuvant. The two following immunizations, in one-weekintervals, were performed using the peptide-conjugate in PBS formulatedwith Freund's incomplete adjuvant. Animals were bled one week after thelast booster dose and collected sera were used for antibody titerdetermination. The titers of specific anti-tau antibodies weredetermined by ELISA, as described in Example 19. Serial dilutions (1:100to 1:102400) of each serum were tested against pathological tauΔ(1-150;392-441)/4R and physiological tau 2N4R, used as a solid phase. FIG.77A-C shows a summary of the results for sera at 1:800 dilution. TheELISA results were statistically evaluated using the Mann-Whitneynon-parametric test. Titers were defined as the reciprocal of thedilution of serum giving one half of maximum OD and summarized in FIG.78.

Immunization of mice with tau peptides TENLKHQPGGGK (SEQ ID NO: 270),KHQPGGG (SEQ ID NO: 271), ENLKHQPGGGKVQIINKKLDLSN VQSKCGS KDNIKHVPGGGS(SEQ ID NO: 275), KHVPGGG (SEQ ID NO: 276), HVPGGG (SEQ ID NO: 277),DNIKHVPGGGSVQIVYKPV (SEQ ID NO: 280), HHKPGGG (SEQ ID NO: 281) andTHVPGGG (SEQ ID NO: 283) induced high levels of tau specific antibodiesin immunized mice. Furthermore, induced antibodies exhibited higheraffinity to pathological tauΔ(1-150; 392-441)/4R than to physiologicaltau 2N4R (FIG. 77A-C). This discrimination was statistically significantfor sera from all immunized animals (SEQ ID NO: 270, p<0.0079; SEQ IDNO: 271, p<0.0052; SEQ ID NO: 275, p<0.0079; SEQ ID NO: 276, p<0.0079;SEQ ID NO: 277, p<0.0379; SEQ ID NO: 280, p<0.0159; SEQ ID NO: 281,p<0.0379, and SEQ ID NO: 283, p<0.0286). Generally, the geometric meanantibody titers to pathological tauΔ(1-150; 392-441)/4R were three- tofive-fold higher than that to physiological tau 2N4R (FIG. 78). As shownin FIG. 78, the highest antibody titers to pathological tau were inducedby tau peptide SEQ ID NO: 275 (GMT 51200), SEQ ID NO: 280 (GMT 51200),SEQ ID NO: 270 (GMT 22286) and SEQ ID NO: 276 (GMT 22286). 5-merpeptides (SEQ ID NOS: 273, 274, 278, 279), which appear to lack thetherapeutic 3D structure, were not able to induce tau specificantibodies in immunized animals. Altogether, these results showed thatthe peptides (SEQ ID NOS: 270, 271, 272, 275, 276, 277, 280, 281 and283) carrying the minimal therapeutic epitopes (therapeutic core units)are immunogenic and induce antibodies with therapeutic potential totarget pathological tau proteins in the brains of Alzheimer's diseasepatients. The aforementioned epitope mapping experiments showed thatpeptide SEQ ID NO: 282 creates a therapeutic 3D structure (e.g., atleast partially mimicks the minimal DC8E8 epitope), nevertheless, it didnot induce a specific antibody response in immunized mice (GMT forpathological tau was 174, FIG. 78).

b) Isotypic Profile:

Vaccination of C57/BL mice with tau peptides SEQ ID NOS: 270, 271, 275,276, 277, 280, 281 and 283 preferentially induced formation of IgG1 andIgG2b antibody isotypes specific to pathological tau. To determine thespecific isotypes of the antibodies produced in response to peptidessera from mice were pooled and diluted from 1:100 to 1:12,800, andtested in duplicates by ELISA (as described in Example 19) againstpathological tauΔ(1-150; 392-441)/4R. To detect mice IgG1, IgG2b, IgG2c,IgG3 and IgM isotypes, anti-mouse subclass specific HRP conjugatedsecondary antibodies were diluted 1:5,000 in PBS (antibodies purchasedfrom Lifespan Biosciences). FIG. 79 shows results for the representative1:800 dilution. The data suggest that the peptides conjugated to KLHinduced a broad spectrum of anti-tau antibody isotypes. In general, thevaccination with peptides generated highest levels of antibody isotypes(IgG1, IgG2b), which are considered to be the high affinity antibodies.The presence of high titers of IgG1 and IgG2b antibodies withpreferential affinity to pathological tau indicated that the immuneresponse induced by the vaccine is directed against pathological tauspecies. The control sera obtained from mock-immunized mice (whichreceived adjuvant alone) were negative (data not shown).

c) Peptides Carrying Therapeutic Epitopes Induce AntibodiesDiscriminating Between Pathological and Physiological Tau:

Real time monitoring of binding events using surface plasmon resonanceenabled measurement of the kinetic rate of association (kON) anddissociation (kOFF) of antibodies from the pooled sera of mice C57/BLimmunized with individual tau peptides TENLKHQPGGGK (SEQ ID NO: 270),KHQPGGG (SEQ ID NO: 271), HQPGGG (SEQ ID NO: 272), HQPGG (SEQ ID NO:273), QPGGG (SEQ ID NO: 274), ENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS(SEQ ID NO: 275), KHVPGGG (SEQ ID NO: 276), HVPGGG (SEQ ID NO: 277),HVPGG (SEQ ID NO: 278), VPGGG (SEQ ID NO: 279), DNIKHVPGGGSVQIVYKPV (SEQID NO: 280), HHKPGGG (SEQ ID NO: 281), HKPGGG (SEQ ID NO: 282) andTHVPGGG (SEQ ID NO: 283). The analysis was done by surface plasmonresonance on BIACORE3000 using a CM5 sensor chip (Biacore AB, Uppsala)as described in Examples 5 and 19. The analysis showed that antibodiesinduced by immunization with the peptides were able to discriminatebetween the recognition of tauΔ(1-150;392-441)/4R and physiological tauisoform 2N4R (FIG. 80). The antibodies induced by immunization withpeptides SEQ ID NOS: 270, 271, 272, 275, 276, 277, 280, 281 and 283,carrying the minimal therapeutic epitopes, exhibited a preferentialaffinity for tauΔ(1-150;392-441)/4R, compared to their affinity for thecorresponding physiological tau 2N4R. This discrimination wasstatistically significant for sera from the peptides: SEQ ID NO: 270(p=0.0392), SEQ ID NO:271 (p=0.0363), SEQ ID NO: 272 (p=0.0022), SEQ IDNO: 276 (p=0.0013), SEQ ID NO: 277 (p=0.0023), SEQ ID NO:280 (p=0.0104),SEQ ID NO:281 (p=0.0123) and SEQ ID NO: 283 (p=0.0011). The obtainedresults are in accordance with previous immunogenicity experimentssummarized in FIG. 77A-C and FIG. 78.

d) Peptide-Induced Antibodies Recognize Pathological Forms of Tau byWestern Blot:

Antibodies induced by immunization of mice C57/BL with individual taupeptides were examined for pathological forms of tau using immunoblotmethod (as described in Example 19). The brain stems of SHR72 rats inthe late stage of neurofibrillary pathology were used for the extractionof insoluble pathological tau proteins. Temporal cortex of human ADbrain (Braak stage VI; obtained from the Netherlands Brain Bank,Netherlands) was used for the extraction of human pathological AD tau.Extracted tau proteins were prepared according to sarcosyl method(Greenberg and Davies 1990). Pooled sera from immunized animals werediluted 1:1000 in PBS and used as a primary antibody. Incubation withprimary antibody was followed by polyclonal rabbit anti-ratimmunoglobulins conjugated to horseradish peroxidase (1:3000; Dako,Glostrup, Denmark). The horseradish peroxidase-conjugated antibodieswere then visualized by chemiluminescence using SuperSignal West PicoChemiluminescence Substrate (Thermo Scientific, Belgium). The signal wasdigitized with LAS3000 CCD imaging system (Fujifilm, Japan). Summarizedresults are provided in FIG. 81. Antibodies elicited by vaccination withpeptides likely creating therapeutic 3D structure of DC8E8 epitope(s)(SEQ ID NOS: 270, 271, 272, 275, 276, 277, 280, 281 and 283) recognizedall pathological forms of tau protein extracted from SHR72 and from ADbrain tissues, including the A68 tau triplet characteristic for AD.Nevertheless, peptide SEQ ID NO: 282 which appears to create atherapeutic 3D structure (competes with tau Δ(1-150;392-441)/4R forbinding to DC8E8), did not induce specific antibody response in thisimmunized mice, thus reactivity was negative. Similarly, 5-mers peptidesthat were not able to induce tau specific antibody response werenegative in this analysis.

e) Peptide-Induced Antibodies Recognize Pathological Tau Proteins in theSections from the Human Alzheimer's Disease Brain Tissues:

Tau-specific antibodies elicited by vaccination of mice C57/BL withindividual peptides were tested on human brain tissue (paraffin blocks)obtained from the Netherlands brain bank. The blocks were cut on amicrotome. Paraffin-sections (8 μm) of the hippocampus-CA1 sector fromAlzheimer's disease brain (Braak stage V) were treated with cold (+4°C.) 99% formic acid for 1 min at room temperature (25° C.). The tissuesections were incubated in blocking solution (5% BSA, 0.3% Triton X-100in 50 nM Tris-HCl) and then overnight with serum diluted 1:1000 inblocking solution. Subsequently, the sections were incubated with abiotinylated secondary antibody (Vectastain Elite ABC Kit, VectorLaboratories) at room temperature for one hour and then reacted withavidin-biotin peroxidase-complex for another one hour (Vectastain EliteABC Kit, Vector Laboratories), both at 25° C. The immunoreaction wasvisualized with peroxidase substrate kit (Vector VIP, Vectorlaboratories, Ca, USA) and counterstained with methyl green (VectorLaboratories). The sections were examined with an Olympus BX71microscope. Immunohistochemical staining (FIG. 82A-C, FIG. 83) suggeststhat the antibodies induced by immunization with peptide SEQ ID NOS:270, 271, 275, 276, 280, 281 and 283 specifically recognizedpathological tau structures, i.e. neurofibrillary tangles in hippocampusof Alzheimer's disease brain tissue. Sera of the animals vaccinated withaforementioned peptides decorated the neurofibrillary pathologyintensively, confirming that the antibodies targeted pathological tauproteins. Antibodies induced by vaccination with peptides SEQ ID NOS:272 and 277 show weaker intensity of staining of pathological taustructures in brain tissues. Peptides that induced lower levels of tauspecific antibody response or did not induce tau specific antibodyresponse (SEQ ID NOS: 273, 274, 278, 279 and 282) were negative in thisanalysis. Sera from mice, which received adjuvant alone, were used asnegative controls. They did not recognize neurofibrillary pathologies(FIG. 82C).

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety. In addition, the following references,which are cited to in the previous paragraphs in a more abbreviatedform, are also incorporated by reference herein in their entirety,including the references cited in such references.

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1-6. (canceled)
 7. An antibody that binds one or more epitopes on tau ina conformationally-specific manner wherein: a) each of the one or moreepitopes is independently selected from HQPGGG (SEQ ID NO: 223), HVPGGG(SEQ ID NO: 154), and HKPGGG (SEQ ID NO: 224) b) zero, one, two, orthree of the epitopes is a linear epitope; and c) one, two, three, orfour of the epitopes is a conformational epitope.
 8. The antibodyaccording to claim 7, wherein the antibody is DC8E8, and wherein DC8E8is an antibody produced by the hybridoma deposited under American TypeCulture Collection Patent Deposit no. PTA-11994.
 9. The antibodyaccording to claim 7, wherein the antibody binds to one or more of thesame epitopes on tau as those bound by DC8E8.
 10. The antibody accordingto claim 7, wherein the antibody competes with monoclonal antibody DC8E8for binding to tau. 11-13. (canceled)
 14. An anti-tau antibodycomprising: a) an antibody light chain variable region comprising: i.QSLLNSRTRKNY (SEQ ID NO: 117) for CDR1; ii. WAS (SEQ ID NO: 118) forCDR2; and iii. KQSFYLRT (SEQ ID NO: 119) for CDR3; and b) an antibodyheavy chain variable region comprising: iv. GYIFTDYVIS (SEQ ID NO: 120)for CDR1 v. IFPRSGST (SEQ ID NO: 121) for CDR2; and vi. ARDYYGTSFAMDY(SEQ ID NO: 122) for CDR1. 15-16. (canceled)
 17. An Fab, Fab′, F(ab′)₂,Fabc, Fv fragment, or any other antigen-binding fragment, or anantigen-binding antibody portion of the antibody of claim
 7. 18. Anantibody that competitively binds to tau with the isolated antibody ofclaim
 14. 19. (canceled)
 20. The antibody according to claim 7, whereinthe antibody is chosen from: a) a monoclonal antibody; b) a polyclonalantibody; c) a recombinant antibody; d) a chimeric antibody; e) ahumanized antibody; f) a human antibody; and g) an antigen-bindingfragment or antigen-binding portion of anyone of (a) through (f). 21.The antibody according to claim 7, wherein the antibody is raised in amammal.
 22. The antibody according to claim 7, wherein the antibody isproduced by a recombinant animal or by a recombinant host cell.
 23. Theantibody according to claim 7, wherein the antibody is detectablylabeled with one or more labeling agents.
 24. The antibody of claim 23,wherein at least one labeling agent is chosen from an enzyme, aradioisotope, a fluorophore, a nuclear magnetic resonance marker, and aheavy metal.
 25. The antibody according to claim 7, further comprisingat least one drug attached to the antibody. 26-28. (canceled)
 29. A cellline expressing the antibody of claim
 7. 30-33. (canceled)
 34. Apharmaceutical composition comprising the antibody according to claim 7and a pharmaceutically acceptable carrier and/or diluent.
 35. (canceled)36. A composition comprising at least one antibody according to claim 7and a diluent or a carrier.
 37. The composition of claim 36, furthercomprising at least one compound or agent selected from a detectablelabel, keyhole limpet hemocyanin, tetanus toxoid or a toxoid derivedfrom other pathogenic bacteria, serum albumins, bovine serum albumin, animmunoglobulin molecule or fragment thereof, thyroglobulin, ovoglobulin,a universal T-cell epitope, a cytokine, a chemokine, IL-1α, IL-1β, IL-2,IL-10, IFN-γ, GM-CSF, MIP1α, MIP1β, and RANTES. 38-40. (canceled)
 41. Amethod of treating or preventing the progression of Alzheimer's diseaseor related tauopathies in a subject, the method comprising administeringto said subject an effective amount of at least one antibody accordingto claim
 7. 42. (canceled)
 43. A method of ameliorating at least one ofthe symptoms associated with Alzheimer's disease or related tauopathiesin a subject, the method comprising administering to said subject aneffective amount of at least one antibody according to claim
 7. 44. Amethod of diagnosing or screening a subject for the presence ofAlzheimer's disease or a related tauopathy in a subject, or fordetermining a subject's risk for developing Alzheimer's disease or arelated tauopathy, the method comprising: a) contacting the subject, ora cell, tissue, organ, fluid, or any other sample of the subject, withan effective amount of at least one antibody according to claim 7; andb) determining the presence of a complex comprising pathological tau andthe antibody, wherein the presence of the complex is diagnostic ofAlzheimer's disease or a related tauopathy associated with the presenceof pathological tau.
 45. A method of monitoring a subject for thepresence, progression, regression, or stabilization of Alzheimer'sdisease or related tauopathies in a subject, or for determining thestage of Alzheimer's disease or related tauopathies in a subject, themethod comprising: a) contacting the subject, or a cell, tissue, organ,fluid, or any other sample of the subject, with an an effective amountof at least one antibody according to claim; and b) determining thepresence of a complex comprising pathological tau and the antibody,wherein the presence of the complex is diagnostic of Alzheimer's diseaseor a related tauopathy associated with the presence of pathological tau.46-55. (canceled)
 56. A peptide, wherein: a) the peptide is a fragmentof tau that is at least 6 amino-acid-residues-long, at least 7amino-acid-residues-long, at least 9 amino-acid-residues-long, at least10 amino-acid-residues-long, at least 12 amino-acid-residues-long, or 30amino-acid-residues-long; and wherein b) the peptide comprises at leastone tau therapeutic epitope.
 57. (canceled)
 58. The peptide of claim 56,wherein the therapeutic epitope is selected from HQPGGG (SEQ ID NO:223), HVPGGG (SEQ ID NO: 154), and HKPGGG (SEQ ID NO: 224).
 59. Thepeptide according to one of claims 56-58, wherein the peptide amino acidsequence is a sequence selected from SEQ ID NOs: 1-4, SEQ ID NOs: 9-101,SEQ ID NOs: 108-112, SEQ ID NO:154, SEQ ID NOs: 223-224, NIKAVPGGGS (SEQID NO: 200), NIKHVPGGGS (SEQ ID NO: 201), IKHVPGGGS (SEQ ID NO: 202),KHVPGGGSV (SEQ ID NO: 203), HVPGGGSVQ (SEQ ID NO: 204), VPGGGSVQ (SEQ IDNO: 205), GWSIHSPGGGSC (SEQ ID NO: 250), SVFQHLPGGGSC (SEQ ID NO: 251),ANIKHVPGGGS (SEQ ID NO: 144), DAIKHVPGGGS (SEQ ID NO: 146), DNAKHVPGGGS(SEQ ID NO: 149), DNIAHVPGGGS (SEQ ID NO: 151), DNIKAVPGGGS (SEQ ID NO:159), DNIKHAPGGGS (SEQ ID NO: 161), and DNIKHVPGGGS (SEQ ID NO: 171).60. (canceled)
 61. A compound comprising the peptide according to claim58 and a moiety.
 62. The compound of claim 61, wherein the moiety isN-terminal, C-terminal, or linked to an internal amino acid of thepeptide, and wherein the moiety is selected from one or more of acysteine residue, phospho group, keyhole limpet hemocyanin, tetanustoxoid or a toxoid derived from other pathogenic bacteria, serumalbumins, bovine serum albumin, an immunoglobulin molecule or fragmentthereof, thyroglobulin, ovoglobulin, a universal T-cell epitope, acytokine, a chemokine, IL-1α, IL-1β, IL-2, IL-10, IFN-γ, GM-CSF, MIP1α,MIP1β, and RANTES.
 63. A pharmaceutical composition comprising thepeptide according to claim 58 and a pharmaceutically acceptable carrier,and/or a diluent, and/or an adjuvant.
 64. A pharmaceutical compositioncomprising the compound according to claim 61 and a pharmaceuticallyacceptable carrier, and/or a diluent, and/or an adjuvant. 65-69.(canceled)
 70. A method of treating or preventing the progression ofAlzheimer's disease or related tauopathies in a subject, the methodcomprising administering to said subject an effective amount of at leastone peptide according to claim 58 and/or at least one compound accordingto claim
 61. 71. (canceled)
 72. A method of ameliorating at least one ofthe symptoms associated with Alzheimer's disease or related tauopathiesin a subject, the method comprising administering to said subject aneffective amount of at least one peptide according to claim 58 and/or atleast one compound according to claim
 61. 73. (canceled)
 74. A method ofproducing an antibody that is able to compete with DC8E8 for binding totau, the method comprising immunizing a subject with a peptide accordingto claim 58 and/or with a compound according to claim
 61. 75. A methodof isolating DC8E8, or isolating an antibody that is able to competewith DC8E8 for binding to tau, the method comprising contacting DC8E8 orthe antibody with a peptide according to claim 58 and/or with a compoundaccording to claim
 61. 76. A method of diagnosing or screening a subjectfor the presence of Alzheimer's disease or related tauopathies in asubject, or for determining a subject's risk for developing Alzheimer'sdisease or related tauopathies, the method comprising: a) contacting thesubject, or a cell, tissue, organ, fluid, or any other sample of thesubject, with an effective amount of at least one antibody according toclaim 7; and b) determining the presence of a complex comprisingpathological tau and the antibody, wherein the presence of the complexis diagnostic of Alzheimer's disease or a related tauopathy associatedwith the presence of pathological tau.
 77. A method of monitoring asubject for the presence, progression, regression, or stabilization ofAlzheimer's disease or a related tauopathy in a subject, or fordetermining the stage of Alzheimer's disease or a related tauopathy in asubject, for the method comprising: a) contacting the subject, or acell, tissue, organ, fluid, or any other sample of the subject, with aneffective amount of at least one antibody according to claim 7; and b)determining the presence of a complex comprising pathological tau andthe antibody, wherein the presence of the complex is diagnostic ofAlzheimer's disease or a related tauopathy associated with the presenceof pathological tau. 78-81. (canceled)
 82. A method of treating orpreventing the progression of Alzheimer's disease or related tauopathiesin a subject, the method comprising administering to said subject aneffective amount of at least one antibody according to claim 7, and/orat least one peptide according to claim 58, and/or at least one compoundaccording to claim 61, in combination with at least one combinationagent chosen from acetylcholinesterase inhibitors, NMDA receptorantagonists, transition metal chelators, growth factors, hormones,non-steroidal anti-inflammatory drugs (NSAID), antioxidants, lipidlowering agents, selective phosphodiesterase inhibitors, inhibitors oftau aggregation, inhibitors of protein kinases, inhibitors of heat shockproteins, anti-amyloid passive and active immunization, anti-amyloidaggregation inhibitors, and secretase inhibitors.
 83. (canceled)
 84. Amethod of ameliorating at least one of the symptoms associated withAlzheimer's disease or related tauopathies in a subject, the methodcomprising administering to said subject an effective amount of at leastone antibody according to one of claims 1-25, and/or at least onepeptide according to claim 7, and/or at least one peptide according toclaim 58, and/or at least one compound according to claim 61, incombination with at least one combination agent chosen fromacetylcholinesterase inhibitors, NMDA receptor antagonists, transitionmetal chelators, growth factors, hormones, non-steroidalanti-inflammatory drugs (NSAID), antioxidants, lipid lowering agents,selective phosphodiesterase inhibitors, inhibitors of tau aggregation,inhibitors of protein kinases, inhibitors of heat shock proteins,anti-amyloid-passive and -active immunization reagents, anti-amyloidaggregation inhibitors, and secretase inhibitors. 85-86. (canceled) 87.A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and/or diluent; and a) an antibody according to claim 7; and/orb) a peptide according to claim 58; and/or c) a compound according toclaim 61; in combination with at least one combination agent chosen fromacetylcholinesterase inhibitors, NMDA receptor antagonists, transitionmetal chelators, growth factors, hormones, non-steroidalanti-inflammatory drugs (NSAID), antioxidants, lipid lowering agents,selective phosphodiesterase inhibitors, inhibitors of tau aggregation,inhibitors of protein kinases, inhibitors of heat shock proteins,anti-amyloid-passive and -active immunization reagents, anti-amyloidaggregation inhibitors, and secretase inhibitors. 88-116. (canceled)